Objective To explore the efficacy and influencing factors of polyethylene glycol interferon α-2b (Peg-IFNα-2b) combined nucleoside analogues (NAs) in the treatment of hepatitis B virus e-antigen (HBeAg)-negative chronic hepatitis B (CHB) patients who had received NAs treatment, and evaluate the correlation of mononucleotide polymorphisms of interleukin-28B and programmed death receptor-1 (PD-1) with interferon treatment response. Methods HBeAg-negative CHB patients who visited Xiangya Hospital of Central South University from January 2020 to December 2022 were analyzed retrospectively. Patients with Peg-IFNα-2b and NAs treatment were as the study group, while those with NAs therapy alone as the control group. Clinical efficacy of two groups of patients at the 12nd, 24th, and 48th weeks of treatment, as well as the persistent response and recurrence at the 72nd week were analyzed. PD-1 and IL-28B single nucleotide polymorphisms were adopted to evaluate the value of HBeAg- negative CHB patients in response to interferon treatment. Results At the 48th week of treatment, the response rate of HBeAg-negative CHB patients in the study group was higher than that in the control group (52.05% [38/73] vs 1.64% [1/61], P < 0.05). Among HBeAg-negative CHB patients in the study group, response rates at 48th week of treatment in patients with baseline HBsAg < 100 IU/mL and HBsAg < 1 000 IU/mL were higher than those with HBsAg≥1 000 IU/mL, respectively (both P < 0.05). Univariate and multivariate analyses showed that in HBeAg-negative CHB patients in the study group, the baseline HBsAg levels (OR=1.004, 95%CI: 1.001-1.006) and HBsAg decline magnitude at the 24th week of treatment (OR=0.111, 95%CI: 0.034-0.362) were influencing factors for the response of interferon treatment combined with NAs (both P < 0.05). The results of single nucleotide polymorphism analysis showed that in HBeAg-negative CHB patients in the study group, the proportion of PD-1 rs10204525 C/T heterozygous mutation in the response population was higher (66.67% vs 16.67%, P < 0.05), while that of IL-28B mutation was not significantly different (P>0.05). Conclusion Combined treatment with Peg-IFNa-2b can achieve higher HBsAg clearance rate and serological conversion rate in HBeAg-negative CHB patients who had received NAs treatment. HBsAg decline magnitude at the 24th week of treatment can better predict the response at the 48th week of treatment. Patients with low baseline HBsAg level and those carrying PD-1 rs10204525C/T heterozygous mutation gene present better therapeutic effect after receiving Peg-IFNa-2b.