Stone fruit processing produces almost 20-30% of waste as seed kernels, which are rich source of valuable compounds like protein that can be used in various food formulations. So, the present study aimed to isolate protein from Meghalayan cherry kernel and study the effect of pH and dephenolization on biochemical, functional, and nutritional characteristics of protein isolate from the Meghalayan cherry kernel. Alkali (pH, 8.5-12.5) extraction and isoelectric precipitation were used to isolate kernel protein. Based on yield, purity, and color, pH 10.5 was most suitable for extracting protein. Dephenoized protein isolate (MCKPI-DP) showed higher protein content (88.81 +/- 0.03%) as compared to natural protein isolate (MCKPI-N) (83.50 +/- 0.01%). The significantly higher solubility, dispersibility, foaming properties, emulsion stability, and capacity were seen in dephenolized protein isolate, which increased with increased pH. Water binding capacity was higher for MCKPI-N than MCKPI-DP and further increased with an increase in pH. Meghalayan cherry kernel protein isolate was a rich source of essential amino acids except tryptophan required by infants and adults (FAO). MCKPI-DP showed better protein efficiency ratio (2.67 +/- 0.03(PER1), 2.49 +/- 0.02(PER2), 2.14 +/- 0.04(PER3)), essential amino acid index (78.82 +/- 0.85), EAA/TAA % (41.76 +/- 0.01), nutritional index (65.82 +/- 0.71), and biological value (74.22 +/- 0.93) indicating the presence of good-quality proteins. As per the FAO standard, the amino acid score showed that all essential amino acids are in excess, thus helping fulfill essential amino acid requirements and being a good protein supplement source in food products. Moreover, this work helps better utilize agro-wastes as low-cost substrates for protein isolation.