MicroRNAs (miRNAs) are essential players of plant defence systems because of their involvement in reprogramming gene expression under adverse environmental conditions including drought and heat, which are considered major players in limiting crop productivity. miR172b-3p was previously determined as a remarkable stress-responsive miRNA in our next-generation sequencing (NGS) analysis in potato. This study aims to understand the functions of miR172b-3p and its target (ERTF RAP2-7-like) under drought, heat, and combined treatments by overexpressing the miR172b-3p in stress-tolerant (Unica) and sensitive (Russet Burbank) potato cultivars. miR172b-3p overexpression in transgenic lines suppressed the ERTF RAP2-7-like expression leading to enhanced carbon fixation efficiency. Meanwhile, the accumulation of hydrogen peroxide (H2O2) was reduced in both cultivars, proving that it is involved in the front-line tolerance mechanism against individual drought, heat, and their combination. In conclusion, our results prove that the stress tolerance could be enhanced by miR172b-3p-mediated negative regulation of ERTF RAP2-7-like gene in potato under drought, heat, and their combination. Our findings represent the first step towards the improvement of tolerance against multiple abiotic stresses in potato.