A kind of new functional polymeric microspheres with strong adsorption ability for creatinine was prepared, and the adsorption mechanism was studied in depth. First of all, the crosslinked copolymer microspheres of 2-hydroxyethyl methacrylate (HEMA) and N-vinylpyrrolidone (NVP) with an average diameter of 180 μm were prepared by suspension copolymerization and denoted as HEMA/NVP, which has biocompatibility. In the suspension copolymerization system, N,N′-methylene biscrylamide (MBA) was used as crosslinking agent, α,α′-azoisobisbutyronitrile (AIBN) was used as initiator, and Span-60 (Sorbitan monooleate) was used as suspension stabilizer. Subsequently, the microspheres HEMA/NVP were chemically modified with 3,5-dinitrobenzoyl chloride (DNBC), and the functional microspheres DNBZ-HEMA/NVP, on which a number of 3,5-dinitrobenzoate (DNBZ) groups were bonded, were obtained. In the modification reaction, triethylamine was used as the agent to capture acid, and reaction could be carried out favourably. The chemical structure and composition of the functional microspheres were characterized with FTIR and chemical analytical method. The adsorption characteristics of DNBZ-HEMA/NVP for creatinine were studied, and the effects of various factors on the adsorption properties of the functional microspheres were investigated. Static adsorption experiments show that the functional microspheres DNBZ-HEMA/NVP have strong adsorption property for creatinine due to the chemical reaction analogous to Jaffe′ reaction between 3,5-dinitrobenzoate on the surface of the microspheres and creatinine. In the aqueous solution, creatinine has two kinds of tautomers, the keto form and the enol form. The tautomer with enol form is easy to loss a proton and produces a negative oxyanion. The negative oxyanions are attracted by the nitrogen atoms with positive electricity on the nitro groups of DNBZ. Finally, a complex reaction takes place and a complex of 1∶1σ is formed between DNBZ of the microspheres and the creatinine molecule. Obviously, the adsorption of the functional microsphere DNBZ-HEMA/NVP for creatinine is attributed to chemical adsorption, furthermore, it is a chemical adsorption driven by electrostatic interaction. The adsorption capacity of DNBZ-HEMA/NVP is enhanced 20 times as compared with that of the unmodified microsphere HEMA/NVP, and the saturated adsorption amount reaches 21 mg/g. Both pH value and salinity influence the adsorption capacity greatly. As pH value of the medium is lower, creatinine exists mainly as keto form, and there are few negative oxyanions, so there is weak electrostatic interaction between the microsphere DNBZ-HEMA/NVP and the creatinine molecule resulting in the poor adsorption ability of the microsphere DNBZ-HEMA/NVP for creatinine and lower adsorption capacity. With the increase of pH value, the equilibrium between the two tautomers of creatinine moves to enol form, and more negative oxyanions will be produced. So the electrostatic interaction is strengthened, resulting in the stronger adsorption ability of the microsphere DNBZ-HEMA/NVP for creatinine and higher adsorption capacity. However, the too high pH value is also disadvantageous to the electrostatic interaction. The experimental results show that as pH=8.5, there is the strongest electrostatic interaction between the microsphere DNBZ-HEMA/NVP and the creatinine molecule, resulting in a maximum adsorption capacity. The ionic strength weakens the lectrostatic interaction, so greater salinity leads to decreasing the adsorption ability of the microsphere DNBZ-HEMA/NVP for creatinine.