Objective To analyze the effect of hemolysis on neonatal neuron specific enolase (NSE) and establish the correction formula of NSE. Methods 50 samples of newborn babies less than 24h old were selected. The upper serum was separated by centrifugation, and repeated freeze-thaw red cell suspension was added. The concentration of NSE in serum was detected by Roche E602 electrochemilescence. The serum hemoglobin (Hb) was detected by instrument method and high ferric cyanide method (HiCN). The NSE correction formula was established respectively. The correlation of NSE concentration changes (ΔNSE) between the instrumental method and HiCN method was analyzed, and the feasibility of the NSE correction formula of the instrumental method was evaluated. Results ΔNSE of 50 hemolytic samples was (35.62±2.17) μg/g Hb. The correlation equation between the change of serum NSE concentration and the change of Hb concentration is Y-axis with ΔNSE as the Y-axis and ΔHBHICN as the x-axis ΔNSE=32.90*Hb whole blood concentration+1.58 (r2=0.991, P＜0.001), YΔNSE=30.97 *HbHICN+1.11 (r2=0.981, P＜0.001). There was a high correlation between NSE results obtained by instrumental method and HiCN method (r2=0.933, P＜0.01), and there was no significant difference between them (P=0.413). Conclusion Hemolytic samples seriously affect the detection results of neonatal NSE. The corrected NSE results can provide reference value for clinical diagnosis.