Ripening as a physico-chemical change is part of a continuous developmental process and hormons play a major role in this processes. The present study was carried out to investigate the effect of external melatonin (0 and 10 μM) injection at the light green stage on the ripening of strawberry fruit. The fruit was sampled for morphological, biochemical, and gene expression analysis during (0, 5, 10, and 15 days after treatment). The results showed a lower accumulation of anthocyanin content was observed in fruits treated with 10 μM. The injection of 10 μM melatonin caused a lower total soluble solid content and fruit color, and higher titratable acidity and softening. The total phenol content was higher in fruit treated with 10 µM melatonin, accompanied by increased PAL enzyme activity and gene expression, increased DPPH scavenging capacity, and higher content of quercetin, gallic, caffeic, and chlorogenic acids. The delay in fruit ripening was associated with suppression of H2O2 level and endogenous ABA accumulation caused by lower expression of NCEDs genes. In general, it is concluded that activating the melatonin ROS scavenging cascade might be responsible for the delayed ripening and development of strawberry fruit. Therefore, our study demonstrates that the exogenous application of 10 μM melatonin can slow the ripening of strawberry fruit.