Objective Non-suicidal self-injury (NSSI) is one of the most common mental health problems and growing public-health issues, coupled with a significant population-level burden among adolescents in both developed and developing countries. We aimed to assess the role of endogenous opioid system-emotion regulation circuitry in NSSI through measurement of plasma beta-endorphin (beta-EP), met-enkephalin (MENK) levels, and determination of psychometric features of Turkish adolescent subjects. Method In this research, we measured plasma beta-EP and MENK levels of 49 adolescents with NSSI and 39 control subjects without NSSI between the ages of 12-18 years. All adolescent subjects were observed in the outpatient clinic, and their clinical and sociodemographic characteristics were examined. All subjects were assessed using the Brief Symptom Inventory (BSI) and Inventory of Statements About Self Injury (ISAS). Results Plasma beta-EP levels were statistically lower in adolescents with NSSI than control group, whereas there was no statistically significant difference in MENK levels. beta-EP levels showed a negative correlation with depression severity. The data obtained from BSI and ISAS were not found to be associated with both beta-EP and MENK levels, while subscale scores exhibited versatile correlations. Conclusion Our findings supported the salient role of beta-EP in NSSI behavior. Also, decreased plasma beta-EP could be assessed as a reliable indicator for NSSI. However, it is possible that measurement of basal plasma levels of neuropeptides might also bring many confounders and could cause bias. Therefore, repeated measurements of plasma-endogenous opioid neuropeptides in a time-dependent manner-concomitant to engage of NSSI behavior-might give more reliable results.