Ovarian cancer has the highest mortality rate among all gynecological cancers, containing complicated heterogeneous histotypes, each with different treatment plans and prognoses. The lack of screening test makes new perspectives for the biomarker of ovarian cancer of great significance. As the main component of extracellular matrix, collagen fibers undergo dynamic remodeling caused by neoplastic activity. Second harmonic generation (SHG) enables label-free, non-destructive imaging of collagen fibers with submicron resolution and deep sectioning. In this study, we developed a new metric named local coverage to quantify morphologically localized distribution of collagen fibers and combined it with overall density to characterize 3D SHG images of collagen fibers from normal, benign and malignant human ovarian biopsies. An overall diagnosis accuracy of 96.3% in distinguishing these tissue types made local and overall density signatures a sensitive biomarker of tumor progression. Quantitative, multi-parametric SHG imaging might serve as a potential screening test tool for ovarian cancer.