L-threitol is a significant intermediate in pharmaceutical synthesis. Previously, we developed a one-pot, two-step multi-enzyme cascade for synthesizing L-threitol from formaldehyde. In this pathway, benzoylformate decarboxylase (BFD) and fructose-6-phosphate aldolase (FSA) catalyze the conversion of formaldehyde into L-erythrulose. This is followed by the reduction of L-erythrulose to L-threitol, driven by L-threitol dehydrogenase (TDH) and a methanol dehydrogenase (MDH)/isopropanol cofactor regeneration system. While this route boasts high atom economy and minimal by-products, the compatibility and optimal concentrations of the enzymes required optimization. [Objective] To systematically optimize the dosages and compatibility of enzymes in the multi-enzyme cascade to enhance both the reaction rate and conversion efficiency. [Methods] The activities of four key enzymes—BFD, FSA, TDH, and MDH—were assessed. Subsequently, factors including enzyme dosage, the cofactor regeneration system, reaction duration, and temperature were optimized step-by-step to improve the system compatibility. [Results] The optimal reaction conditions were determined as follows: enzyme dosages of BFD, FSA, TDH, and MDH being 10, 1, 1, and 8 mg/mL, respectively. Notably, the dosages of FSA and TDH were reduced by 87% and 67%, respectively, compared with pre-optimization levels. Other optimal parameters included a NAD⁺ concentration of 2 mmol/L, a reaction temperature of 30 ℃, and reaction duration of 8 h (representing a 60% decrease from that of the original system). Under these conditions, the maximum L-threitol concentration reached 166.76 mmol/L, with a yield of 89%. In a scale-up experiment, the L-threitol yield remained at 80%, representing a 43% increase compared with the pre-optimization level. [Conclusion] By systematically optimizing enzyme compatibility and reaction conditions, this study significantly reduces the enzyme dosages and reaction duration for converting formaldehyde to L-threitol. Simultaneously, it substantially improves the production efficiency and yield, establishing a robust foundation for the enzymatic synthesis of L-threitol from formaldehyde.