Women with polycystic ovary syndrome (PCOS) are reported to have different levels of prolactin (PRL) compared to women without PCOS. This study aimed to evaluate the PRL levels in women with PCOS, compared to the control group, before and after adjustment for potential confounders. Using a logical combination of keywords, a comprehensive search was carried out in PubMed and Web of Science, from inception to 30 August 2022. Weighted mean differences (WMDs) with corresponding 95% CIs in PRL levels were employed with a random-effects model. I2 was applied to evaluate heterogeneity among studies. A meta-regression analysis and subgroup analysis were conducted to explore heterogeneity sources. Publication bias was assessed by the Egger test. Thirty-two studies, measuring PRL levels in 8551 PCOS patients according to the Rotterdam criteria and 13,737 controls, were included in the meta-analysis. Pooled effect size suggested that the overall weighted mean difference (WMD) of PRL level was significantly higher in women with PCOS, compared to controls (WMD = 1.01, 95% CI: 0.04–1.98, p = 0.040). The result of meta-regression adjusted for age, BMI, and the continent of origin, revealed no confounding effect on results. Sub-group analysis of PRL levels according to the continent of origin showed significantly higher PRL levels among Eurasian PCOS patients compared to the control; this difference was not statistically significant in the subgroups of women from Asia, Europe, and South America. In conclusion, PRL levels in patients who were diagnosed according to the Rotterdam criteria were significantly higher than non-PCOS participants. Slightly higher levels of PRL could be presented as a diagnostic feature of PCOS.