Background Glue sealing has become an alternative option for occlusion of enterocutaneous fistula (ECF) because of it minimal invasiveness and simplicity. This study aimed to determine efficacy and safety of autologous, platelet-rich fibrin glue (PRFG) in promoting closure of ECFs. Methods This was a nonrandomized cohort study, recruiting patients who had low-output ECFs (<200 mL/24 h). Beside standard of care, patients were assigned to either the PRFG or control group. Clinical efficacy and safety were determined prospectively. Moreover, a well-balanced subcohort was generated by propensity score (PS) matching. Unadjusted and adjusted Cox proportional hazard models were employed to determine hazard ratios (HRs) of ECF closure in both cohorts. Results From January 2008 to January 2012, 145 patients were enrolled initially, with 70 in the control group and 75 in the PRFG-treated group. Compared with the control group, patients in the PRFG group had lesser median time of fistula closure (7 vs 23 days; P = .0010). In addition, PRFG healed more fistulas within the first 28 days (77% vs 57%; P = .0127). For all fistulas included, PRFG-treated fistulas were 3.13 (95% confidence interval [CI], 1.82–5.36) times more likely to achieve closure than those with the non-PRFG approach in the adjusted Cox model. In a PS-matched cohort with 28 paired fistulas, HRs were 3.41 (95% CI, 1.91–6.07) for all fistulas regardless of location. No adverse events related to glue applications were observed. Conclusion Autologous PRFG seems to be safe and effective in the treatment of low-output ECFs, and can lessen closure times and promote closure rates.