Mechanochemistry, a sustainable and efficient approach, is now used to encapsulate biocatalysts like enzymes within MOFs (enzyme@MOFs). Yet, achieving ultrafast, eco-friendly, and high-yield synthesis of enzyme@MOFs, with potential for mitigating biocatalyst and industrial MOF production improvement, remains challenging. In this study, we illustrated the enzyme one-pot mechanical encapsulation within zeolitic imidazolate framework-90 (ZIF-90), a MOF subfamily. A substantial yield (ca. 80%) was achieved in only 10 s at 8 Hz or lower frequencies, assisted by a small amount (∼100 μl) of Tris buffer solution. The encapsulated enzyme catalase (CAT), responsible for decomposing hydrogen peroxide, maintains its bioactivity after mechanical treatment. Additionally, the enzyme gains protection from digestion (e.g., proteinase K) due to the size-sheltering effect. Simulations of energy transfer and experimental findings reveal that a buffer solution containing primary amines affects linker deprotonation, thereby facilitating the rapid formation of a well-defined 3D structure of enzyme@ZIF-90 crystals during ultrafast milling reactions. We further demonstrated our mechanical approach by encapsulating bovine serum albumin and Escherichia coli in ZIF-90, highlighting the versatility of this method for crafting MOF biocomposites across diverse industrial applications.