Background: Serum tumor markers are extensively used for assisting cancer diagnoses, therapeutic monitoring, and prognostication in clinical practice. Aim: This study aimed to evaluate the levels of six selected serum tumor markers and their association with gender and age risk factors in a Libyan population. Methods: Serum samples were obtained from each individual involved in this study and the level of CEA, CA125, CA15‑3, CA19‑9, AFP and PSA tumor markers were quantified by an electrochemiluminescence immunoassay analyzer. Results: Among the 2072 participants, 18.8% of individuals showed abnormal serum levels of tumor markers and the mean serum levels of all measured tumor markers were higher than their normal ranges used in this study with higher levels of CEA and CA125 were reported in females when compared to males. CEA, CA19‑9, PSA and fPSA levels increased with age while AFP levels were high only in the 1st decade of age. On the other hand, CA125 and CA15‑3 levels did not vary with age. Conclusion: This study has documented the serum levels of six tumor markers and identified significant gender and age differences in their levels in a Libyan population.