ABSTRACT: This study investigates an eco-friendly approach to cotton fabric coloration using a natural dye extracted from achiote tree seeds (Bixa orellana) combined with a sustainable, bio-mordant. The primary objective is to extract and characterize plant based biodegradable natural dye and mordant that can be used as alternative option for toxic synthetic colorants and hazardous metallic mordants in dyeing of cotton. Through optimized alkaline extraction at 85°C for 2 hours, a maximum crude dye yield of 25 % was achieved. Subsequent exhaust dyeing trials demonstrated optimum color strength (K/S) of 5.18. The protein-mordanted cotton substrates exhibited reliable fastness characteristics, presenting a sustainable, non-hazardous alternative for eco-conscious textile product development. Keywords: Glycine max protein mordant, Bixa orellana seed, color strength, color fastness