Pseudomonas aeruginosa preparation (PAP) has shown activity in inhibiting the metastasis of cancer and can improve the immune function of cancer patients during chemotherapy. This study aimed to investigate whether PAP can improves the efficacy of chemotherapy in patients with advanced non-small-cell lung cancer (NSCLC). A total of 72 subjects with stage IIIB/IV NSCLC were randomized into PAP arm (n = 36) or control arm (n = 36) at a 1:1 ratio. Subjects had vinorelbine and cisplatin combined with 0.5 ml PAP/placebo on d1, followed by 1 ml PAP/placebo injected subcutaneously three times a week. The primary end point was the objective response rate (ORR) and secondary endpoints were time to progression (TTP), overall survival (OS), safety and quality of life (QOL). Sixty-six patients were included in intent-to-treat analysis. After two cycles of treatment, there was a borderline statistically significant improvement in ORR of PAP arm (46.88 vs. 23.53 %, P = 0.0532), and after four cycles of treatment, the ORR in PAP arm was 31.25 versus 14.71 % in control arm (P = 0.1110). Median TTP and OS were 160 and 454 days in PAP arm, 196 and 388 days in control arm, P = 0.4609 and 0.6587, respectively. The 1-year survival rate in PAP and control arms was 53.55 and 50.15 %, respectively. PAP did not result in increased toxicity and or had negative impact on QOL. The results demonstrate the therapeutic potential of PAP for advanced NSCLC. PAP can be used with chemotherapy to improve the response rate. Long-term follow-up might help define whether the combination therapy can result in survival benefit.