Exploration of the exposure of preconception couples to per- and polyfluoroalkyl substances (PFAS), as well as the most important influencing factors, promotes the understanding of the joint effects of parental exposure on reproductive health. In the present study, a total of 938 preconception couples recruited through the Shanghai Birth Cohort were investigated for the variations of PFAS exposure and contributing factors within and between couples. While linear perfluorooctanoic acid (n-PFOA, median 20.4 ng/mL) and linear perfluorooctanesulfonic acid (n-PFOS, 12.1 ng/mL) remained dominant in plasma, emerging PFAS, particularly 6:2 chlorinated polyfluorinated ether sulfonate (10.5 ng/mL), 6:2 polyfluoroalkyl phosphate diester (0.41 ng/mL), and branched PFOS or PFOA isomers, were also frequently detected. Although individual PFAS were generally correlated within couples, gender differences significantly existed in the concentrations of most individual PFAS and isomer profiles of PFOS and PFOA. Men generally exhibited higher plasma concentrations than their partners, likely reflecting gender-specific elimination pathway and kinetics. Couple-based PFAS exposure also varied greatly. After adjustment for individual factors, several household factors, including annual household income, dwelling floor type, drinking water source, and living near farmlands, were found to be associated with couple-based PFAS exposure. Our study constitutes one of the few studies addressing couple-based exposure to PFAS and lays a solid ground for further assessment of the impacts of parental exposure on reproductive health.