To analyze the expression levels of elastase (NE) and miR-551b in prostatitis massage fluid (EPS) of infertility men with prostatitis, and to study their diagnostic value. Methods: Between July 2017 and May 2019, 60 infertility men with prostatitis were selected in observation group, and 60 healthy male volunteers were selected in control group during the same period. The expressions levels of NE and miR-551b in EPS of man were compared between the two groups. The correlation between NE level and miR-551b level was analyzed. And the diagnostic efficacy and the area under ROC curve (AUC) of the levels of NE and miR-551b of infertility men with prostatitis were also analyzed. Results: The NE expression level of men in the observation group was 1472.25 (812.23, 1735.65), which was significant higher than that ［156.38 (76.35, 226.52)］ of men in the control group. The expression level of mir-551b of men in the observation group was 5.13 (2.34, 13.47), which was significant lower than that ［22.31 (8.64, 42.23)］ of men in the control group (P<0.05). Spearman rank correlation analysis showed that NE level was negative correlated with the miR-551b level of men in the observation group (r=-0.874, P<0.05). The sensitivity, the specificity, and the accuracy of the levels of NE and miR-551b for combined diagnosing prostatitis of infertility men were 79.2%, 81.5%, and 84.3%, respectively, which were all significant higher than those of diagnosing by NE level or miR-551b level only. The AUC of NE and miR-551b for combined diagnosing infertility of man with prostatitis was also significant higher than that of diagnosing by NE level or miR-551b level only (P<0.05). Conclusion: The combined of NE and miR-551b levels in EPS has better value for diagnosing infertility of men with prostatitis, which is expected to be molecular markers for diagnosing infertility of men with prostatitis.