Objective: To evaluate the relationship between serum prostate-specific antigen (PSA) level and total cholesterol, fasting blood glucose, and neutrophillymphocyte ratio (NLR) in men without prostate cancer. Materials and Methods: Between 2010 and 2017, 2631 male participants aged 50-70 years with a serum PSA level of 0-10 ng/mL were included from a population of 4643 healthy males who participated in a health screening program conducted by the İzmir Metropolitan Municipality Eşrefpaşa Hospital in the district villages of İzmir. Participants' serum PSA, fasting blood glucose, total cholesterol levels, and NLR were retrospectively assessed. Participants were grouped as those with high and low serum PSA levels, high and low glucose levels, and high and low cholesterol levels. Differences between the groups in terms of serum PSA levels, serum total cholesterol, glucose, and NLR were analyzed. Results: The mean age of the participants was 60.2±5.4 years and the mean serum PSA level was 1.28±1.20 ng/mL. The mean PSA value was higher in the high cholesterol group (1.36±1.33 vs 1.19±1.02, p<0.001) compared to the low cholesterol group, and the mean PSA value in the high glucose group was lower than in the low glucose group (1.08±0.86 vs 1.32±1.25, p<0.001). Compared to the normal PSA group, the high PSA group had higher mean cholesterol level (208.6±39.9 vs 203.3±41.8, p<0.001) and NLR (2.17±1.00 vs 2.06±0.89, p=0.039), but lower glucose level (108.5±32.5 vs 117.2±51.0, p=0.004). Serum PSA level was positively correlated with total cholesterol and NLR (r=0.074 and p<0.001, r=0.050 and p=0.011), and negatively with glucose level (r=-0.084 and p<0.001). Conclusion: Evaluation of total cholesterol, fasting glucose, and NLR, which may be associated with serum PSA levels, may help urologists when investigating elevated serum PSA levels in asymptomatic men aged 50-70 years who have not been diagnosed with prostate cancer.