Cancer is the second leading cause of death in the world. Reports on the effect of Trimethylamine-N-oxide (TAMO), a small amine oxide generated by gut microbial metabolism of choline, betaine, and carnitine, on cancer are inconsistent. Therefore, this systematic review and meta-analysis summarize the effect of TAMO on cancer incidence. A systematic search was conducted in PubMed, Scopus, Web of Science, and Embase. Data were pooled using the random-effects method and were expressed as weighted mean difference (WMD) and 95% confidence intervals (CI). The pooled results of 16 studies, including 5930 participants, showed that the association between TMAO levels and cancer incidence is insignificant (Odds Ratio: 0.97, 95% CI: (0.64, 1.46), P-value = 0.871). Subgroup analysis showed that urinary TMAO levels were negatively associated with cancer incidence; in contrast, a direct and positive association was observed between serum TMAO levels and cancer incidence. However, “gender” and the “TMAO measuring method” were the potential sources of discrepancies. Meta-regression analysis did not reveal any significant association between duration of studies, age, female ratio, subjects-control, and subjects-case. The present study demonstrates that serum TAMO levels were insignificantly associated with cancer incidence.