Objective To analyze alterations in pelvic floor structure among patients with pelvic floor dysfunction （PFD） using perineal real-time three-dimensional ultrasound and to investigate the associations with serum levels of relaxin， connective tissue growth factor （CTGF）， and soluble microfibril-associated protein 4 （sMFAP4）. Methods The clinical data of 102 puerperae diagnosed with PFD at 42 ~ 60 days postpartum between August 2022 and July 2025 were retrospectively reviewed （PFD group）. Based on the severity of pelvic organ prolapse， the patients were further classified into a mild （Stage Ⅰ） subgroup （n = 43） and a moderate （Stage Ⅱ） subgroup （n = 59）. Additionally， 32 puerperae without PFD who underwent routine postpartum examinations during the same period were enrolled as the control group. Fasting peripheral cubital venous blood samples were collected on the day of examination to measure serum levels of relaxin， CTGF， and sMFAP4， and perineal real-time three-dimensional ultrasound was performed to evaluate pelvic floor structural parameters. The differences in these ultrasound parameters and serum biomarker levels were compared across the three groups. Pearson correlation analysis was employed to assess the association between perineal real-time three-dimensional ultrasound parameters and serum levels of relaxin， CTGF， and sMFAP4 in the PFD group. To account for potential confounding factors， multivariate linear regression analysis was subsequently performed to further examine these associations. Results There were no significant differences in ultrasound-based pelvic floor parameters， such as levator hiatus area at rest， among the three groups （P > 0.05）. However， the levator hiatus area during maximal Valsalva maneuver， as well as serum levels of relaxin and CTGF， were significantly higher in the PFD grade Ⅱ subgroup compared to both the PFD grade Ⅰ subgroup and the control group （P < 0.05）. Moreover， these parameters were also elevated in the PFD grade Ⅰ subgroup relative to the control group （P < 0.05）. Serum sMFAP4 levels were significantly lower in the PFD Ⅱ subgroup compared to both the PFD I subgroup and the control group （P < 0.05）， and were also lower in the PFD I subgroup than in the control group （P < 0.05）. Pearson correlation analysis showed that in the PFD group， the levator hiatus area during maximum Valsalva maneuver was significantly positively correlated with serum relaxin and CTGF levels （r = 0.655， r = 0.721， P < 0.05）， and significantly negatively correlated with serum sMFAP4 （r = -0.493， P < 0.05）. Multivariate linear regression analysis revealed that， after adjusting for potential confounding factors?including age， gestational age at delivery， time from postpartum to examination， body mass index， parity （primipara vs. multipara）， and neonatal birth weight—the levator hiatus area during maximal Valsalva maneuver remained significantly positively associated with serum relaxin （β = 0.312， 95%CI： 0.070 ~ 0.596， P < 0.05） and CTGF （β = 0.972， 95% CI： 0.571 ~ 1.682， P < 0.05）， and significantly negatively correlated with sMFAP4 （β = -0.352， 95% CI： -0.614 ~ -0.102， P < 0.05）. Conclusion Perineal real-time three-dimensional ultrasound imaging of puerperal women with PFD revealed a significantly increased area of the levator hiatus during maximal Valsalva maneuver， which was correlated with serum levels of relaxin， CTGF， and sMFAP4， offering potential insights for clinical management strategies.