Diabetes mellitus is a kind of highly prevalent chronic disease in the world. The intervention measures on the risk factors of prediabetes contribute to control and reduce the occurrence of diabetes. This study aimed to investigate the correlation between proinsulin (PI), true insulin (TI), PI/TI, 25(OH) D3, waist circumference (WC), and risk of prediabetes. Methods In this cross-sectional study, 1662 subjects including 615 prediabetes and 1047 non-prediabetes were recruited. Spearman's correlation analysis was used to explore the association of PI, TI, PI/TI, 25(OH) D3, and waist circumference with prediabetes. Odds ratios (OR) and 95% confidence intervals (CI) were calculated by logistic regression. Receiver-Operator Characteristic (ROC) curve was used to evaluate the risk of prediabetes. Results Our study showed that FPI, 2hPI, FTI, 2hTI, FPI/FTI, and WC could enhance the risk of prediabetes (OR 1.034; OR 1.007; OR 1.005; OR 1.002; OR 3.577, OR 1.053, respectively; all p< 0.001). Stratified analyses indicated that FPI/FTI associated with an increased risk of prediabetes in men (OR 2.080, p = 0.042). FTI have a weak association with prediabetes risk in men and women (OR 0.987, p = 0.001; OR 0.994, p = 0.004, respectively). 2hPI could decrease prediabetes in women (OR 0.995, p = 0.037). Interesting, the sensitivity (86.0%) and AUC (0.942, p< 0.001) of combination (FPI+FTI+2hPI+2hTI+25(OH) D3+WC) were higher than the diagnostic value of these alone diagnoses. The optimal cutoff point of FPI, FTI, 2hPI, 2hTI, 25(OH) D3, and WC for indicating prediabetes were 15.