β2-microglobulin (β2m) is an essential component of major histocompatibility complex class I (MHC I) and plays a key role in antigen presentation. In vitro reconstitution of peptide–MHC I complexes typically requires co-refolding of β2m with the heavy chain. However, recombinant β2m is commonly expressed as insoluble inclusion bodies in Escherichia coli, limiting efficient large-scale production. Here, we demonstrate that the E. coli Shuffle T7 strain enables soluble expression of β2m, achieving yields of 30–40 mg/L in LB medium—over tenfold higher than conventional periplasmic expression methods. The purified β2m efficiently supports refolding with MHC I heavy chains, resulting in functional peptide–MHC I complexes. This streamlined production strategy provides a practical and scalable platform for generating functional β2m, facilitating immunological and structural studies.