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The reverse transcriptase of Moloney murine leukemia virus (MMLV) is an enzyme that synthesizes DNA from an RNA template. Among reverse transcriptases, this enzyme is currently the most commonly used in molecular biology and diagnostics. Since its discovery, this viral protein has been extensively studied, shedding light on its structural and functional characteristics, and offering opportunities to optimize its catalytic performance for biotechnological applications. The review describes the structural motifs of the reverse transcriptase of MMLV, the key amino acids in enzymatic catalysis, and the enzyme’s properties such as processivity, fidelity, and thermal stability. This review reports the optimizations made to improve these parameters, which enabled the modified enzymes to be integrated in the molecular biology tests used in the laboratory on a daily basis.