In order to investigate the metabolic characteristics of human follicular fluid (FF) and to reveal potential metabolic predictors of follicular development (FD) with clinical implications, we analyzed a total of 452 samples based on a two-stage study design. In the first stage, FF samples from both large follicles (LFs) and matched-small follicles (SFs) of 26 participants were analyzed with wide-spectrum targeted metabolomics. The metabolic signatures were described by multi-omics integration technology including metabolomic data and transcriptomic data. In the second stage, the potential biomarkers of FD were verified using enzyme-linked immunoassay with FF and blood serum from an independent 200 participants. We describe the FF metabolic signatures from ovarian follicles of different developmental stages. Lysophosphatidylcholine (LPC) can be used as a biomarker of FD and ovarian sensitivity, advancing the knowledge of metabolic regulation during FD and offering potential detection and therapeutic targets for follicle and oocyte health improvements in humans.