To promote the development and utilization of Acer truncatum seed oil (a novel food resource) and its key component, nervonic acid, this study investigated the enrichment of nervonic acid in the seed oil through low-temperature crystallization combined with urea complexation. The seed oil was obtained by ultrasonic-assisted extraction, and its fatty acid composition was analyzed by GC-MS. The results revealed the fatty acids (content) including linoleic acid (36.00%), oleic acid (24.57%), erucic acid (17.15%), cis-11-eicosenoic acid (7.64%), nervonic acid (4.98%), palmitic acid (3.62%), and other fatty acids (6.04%). Single factor experiment and orthogonal test were conducted to optimize the saponification conditions of A. truncatum seed oil. The optimal parameters were determined as follows: saponification temperature of 80 °C, saponification time of 80 min, and saponification solution concentration of 1.5 mol/L. Under these conditions, the yield of mixed fatty acids reached 73.52%. Further, the combined method of low-temperature crystallization and urea complexation was used to enrich nervonic acid and optimize the process conditions. Low-temperature crystallization was performed under the conditions of a material-to-liquid ratio of 1:3, crystallization temperature of -20 °C, and crystallization time of 10 h, followed by urea complexation under the conditions of a mixed fatty acid/urea/methanol (W/W/V) ratio of 1:3:9 and a complexation temperature of -10 °C. Under these optimized conditions, the nervonic acid content increased to 20.21%, representing a nearly fourfold enhancement compared to its original concentration in the crude seed oil. This study provides a novel methodology for enriching nervonic acid from A. truncatum seed oil, offering significant potential for advancing the industrial applications of the products derived to this plant.