Abstract: The polypeptide-rich liquid extracted from alkaline thermal hydrolysis (ATH) sludge has proven to be non-toxic and usable in agricultural production, resulting in a significant increase in crop growth. To explore the effect of ATH hydrolysate on nitrogen uptake and nitrogen metabolism regulation mechanism of Brassica chinensis, B. chinensis was flushed with ATH hydrolysate (0 mg·kg⁻¹, 20.19 mg·kg⁻¹, 40.38 mg·kg⁻¹, 60.57 mg·kg⁻¹, and 80.76 mg·kg⁻¹) treatments, with no nitrogen fertilizer as the control in a pot experiment. The results showed that as the application rate of flushed ATH hydrolysate increased, all indices first increased and then decreased. When the ATH hydrolysate was flushed at 40.38 mg·kg⁻¹, the nitrogen accumulation, yield, and quality of B. chinensis were higher than those of the control after harvest, and the nitrate content was the lowest. By fitting the nitrogen uptake and yield of B. chinensis, the optimal application amount of ATH hydrolysate was determined to be 121.48−127.59 kg·hm⁻². Under the condition of 40.38 mg·kg⁻¹ for potted B. chinensis, nitrate reductase (NR), nitrite reductase (NiR), glutamate dehydrogenase (GDH), glutamate synthase (GOGAT), and glutamine synthetase (GS) activities could maintain high activity. In the 2nd, 4th, and 6th weeks after final singling of seedlings, the NR activity increased by 56.56%−183.43%, 16.55%−150.36% and 7.86%−293.25%, NiR activity increased by 24.70%−348.17%, 1.06%−71.24% and 7.62%−286.59%, GDH activity increased by 9.91%−149.21%, 37.52%−308.35% and 16.08%−123.12%, GS activity increased by 4.13%−17.82%, 5.23%−122.27% and 9.91%−121.21%, and GOGAT activity increased by 31.31%−288.16%, 9.63%−351.69% and 28.45%−1274.32%. Redundancy analysis showed that GOGAT in B. chinensis was the main factor determining the yield, nitrogen utilization rate, and nitrogen absorption rate, with a significant positive correlation. The ATH hydrolysate solution had a significant influence on the yield of B. chinensis and the activities of enzymes related to nitrogen absorption and transformation under different flushing rates. An appropriate amount of ATH hydrolysate increases the activity of nitrogen-related invertase in B. chinensis, thereby improving the absorption and utilization of nitrogen and yield. At the same time, it also showed that ATH solution can be used as a new type of fertilizer, which not only solves the problem of sludge but also improves the yield and nutrient absorption of B. chinensis.