Mycotoxins are a class of exogenous metabolites that are major contributors to foodborne diseases and pose a potential threat to human health. However, little attention has been paid to trace mycotoxin co-exposure situations in vivo. To address this, we devised a novel analytical strategy, both highly sensitive and comprehensive, for quantifying 67 mycotoxins in human plasma samples. This method employs isotope dilution mass spectrometry (IDMS) for approximately 40% of the analytes and utilizes internal standard quantification for the rest. The mycotoxins were classified into three categories according to their physicochemical properties, facilitating the optimization of extraction and detection parameters to improve analytical performance. The lowest limits of detection and quantitation were 0.001-0.5 μg/L and 0.002-1 μg/L, respectively, the intra-day precision ranged from 1.8% to 11.9% RSD, and the intra-day trueness ranged from 82.7-116.6% for all mycotoxins except Ecl, DH-LYS, PCA, and EnA (66.4-129.8%), showing good analytical performance of the method for biomonitoring. A total of 40 mycotoxins (including 24 emerging mycotoxins) were detected in 184 plasma samples (89 from infertile males and 95 from healthy males) using the proposed method, emphasizing the widespread exposure of humans to both traditional and emerging mycotoxins. The most frequently detected mycotoxins were ochratoxin A, ochratoxin B, enniatin B, and citrinin. The incidence of exposure to multiple mycotoxins was significantly higher in infertile males than in healthy subjects, particularly levels of ochratoxin A, ochratoxin B, and citrinin, which were significantly increased. It is necessary to carry out more extensive biological monitoring to provide data support for further study of the relationship between mycotoxins and male infertility.