Glucosamine (GlcN, 2-amino-2-deoxy-D-glucose) is an amino sugar and a key structural component of cartilage, commonly produced from animal sources, microbial fermentation, and fungi. Owing to its extensive applications in osteoarthritis treatment, dietary supplements, and cosmetic formulations, achieving high glucosamine purity is essential. This study investigates the purification of glucosamine using cooling crystallization, a method that induces supersaturation through controlled temperature reduction. Optimal crystallization was achieved from a saturated feed solution at 0 °C under 500 rpm agitation for 6 h. SEM analysis showed that the obtained crystals exhibited smooth and compact morphology. FTIR analysis confirmed the presence of characteristic functional groups, including O–H (3283 cm⁻¹), N–H (1657.97 cm⁻¹), and C–N (1154.28 cm⁻¹). HPLC analysis yielded a retention time of 1.962 min, indicating successful purification. These results demonstrate that cooling crystallization is an effective and straightforward technique for producing high-quality glucosamine suitable for pharmaceutical and cosmetic applications.