Abstract: To develop an ultrafiltration process for separating α-lactalbumin (α-La) and achieve high-value utilization of by-products, this study employed a small-scale tangential flow ultrafiltration equipment to optimize the process parameters for enriching α-La from whey protein concentrate. Additionally, the study investigated the heat treatment modification of the by-product after α-La separation. The results showed that the optimal conditions for α-La separation were as follows: Feed pH of 6.5, ultrafiltration membrane molecular weight cutoff of 100 kDa, volume concentration factor of 4, operating pressure of 0.3 MPa, and 5 diafiltration cycles. Under these conditions, the ratio of α-La to β-lactoglobulin (β-Lg) in the permeate increased to over 9, a 23-fold improvement compared to the whey protein concentrate. The foaming properties of the β-Lg-rich retentate, remaining after α-La separation, were optimal under heat treatment at 60 ℃ for 10 minutes, with foam ability and foam stability reaching 18.1% and 69.9%, respectively. In contrast, the emulsifying properties were optimal at 80 ℃ for 10 minutes, with emulsifying activity index and emulsifying stability index reaching 77.0 m²/g and 82.8%, respectively. Overall, this study established an optimal membrane separation process for enriching α-La and enhanced the functional properties of the by-product retentate through heat treatment, providing a valuable technical solution for the industrial production of α-La and the comprehensive utilization of by-products.