Studies have demonstrated the worldwide presence of bisphenol A (BPA) and its toxic effects on human health. BPA may exist as several structural isomers, which are byproducts in industrial BPA production. However, nearly nothing is known about the occurrence of BPA isomers in human blood and the partitioning of BPA metabolites between human serum and whole blood. In this study, BPA, BPA-sulfate (BPA-S), and BPA-glucuronide (BPA-G) were quantified in 144 pairs of serum and whole blood samples from Chinese participants. BPA was detected in 115 serum and 121 whole blood samples, with mean concentrations of 0.53 and 0.88 ng/mL, respectively. A structural isomer of BPA, tentatively termed B₁-BPA, was identified for the first time, and measurable in 53% and 57% of serum (<LOD–1.9 ng/mL) and whole blood (<LOD–1.4 ng/mL) samples, respectively. BPA-S was the predominant BPA metabolite (mean 2.3 and 1.4 ng/mL, respectively), significantly higher (p < 0.01) than BPA-G (1.3 and 0.64 ng/mL) in both serum and whole blood. The calculated partitioning coefficients between serum and whole blood were the highest for B₁-BPA (mean ± SD, 1.8 ± 0.25), followed by BPA-S (1.6 ± 0.36), BPA-G (1.4 ± 0.37), and BPA (1.3 ± 0.39), indicating their preferential enrichment in the serum fraction. Overall, this study first identifies a BPA isomer, which has not been previously reported in any environmental or human samples. Measuring BPA isomers in human serum and whole blood is critical for accurate human BPA exposure risk assessment.