Exposure to pesticides can pose a series of advance effects on human health. However, the exposure levels and health implications of the current use pesticides and their metabolites in both men and pregnant women remain unclear. In this study, an analytical method was developed to quantify fungicides, neonicotinoid insecticides, triazine herbicides, and their metabolites in the human serum. Fifty of the 73 target pesticides and metabolites were detected in the human serum of men and pregnant women from Wuxi, China, which included 11 triazine herbicides and metabolites, 17 neonicotinoid insecticides and metabolites, and 22 fungicides. Fungicides had the highest cumulative concentration (49.5 ng/mL), followed by neonicotinoid insecticides and metabolites (6.38 ng/mL), and triazine herbicides and metabolites (5.10 ng/mL). Moreover, the estimated daily intake (EDI) of fungicides was 10.4 and 12.7 times higher than that of triazine herbicides (included their metabolites) and neonicotinoid insecticides (included their metabolites), respectively. Of the three categories of pesticides, exposure to fungicides contributed to the highest exposure risk within the hazard quotient in the range of 5.1 × 10 -0.17. Correlation analysis revealed that the pesticide exposure levels in human serum were correlated with their maximum residue levels in vegetables and fruits. Pesticide exposure has also been correlated with the weight and Body Mass Index (BMI) of humans based on structural equation modeling. This study provides new insights into the exposure of men and pregnant women to a cocktail of fungicides, neonicotinoid insecticides, triazine herbicides and their metabolites.