The invention relates to a formulation for a biocatalyst for modifying and improving petroleum fuel oil and a production method thereof. The formulation comprises the main components: 1, vegetable fat; 2, methanol; 3, candida rugosa lipase; 4, candida lipase; 5, horse radish peroxidase; 6, hydrogen peroxidase; 7, polyethylene glycol 1,000 to 5,000 (PEG for short); and 8, single methoxy polyethylene glycol 5,000 to 10,000 (MPEG for short). The production method comprises the following production processes and usage amount of raw materials is as follows, according to 1,000 liters of biocatalyst product produced once for modifying and improving the petroleum fuel oil: 1, the first step: using 'biological generator' equipment to produce 'fatty acid methyl ester or ethyl ester'; 2, the second step: taking 10 to 12 grams of hydrogen peroxidase powder and 5 grams of single methoxy polyethylene glycol 5,000 powder, adding 10 milliliters of pure distilled water into the mixture for mixing and preparing; and keeping stand the mixture for 30 minutes; 3, the third step: taking 12 to 15 grams of horse radish peroxidase powder and 8 grams of the single methoxy polyethylene glycol 5,000 powder, adding 12 milliliters of pure distilled water into the mixture for mixing; and keeping stand the mixture for 30 minutes; 4, the fourth step: mixing hydrogen peroxidase preparation and horseradish peroxidase preparation in turn in an organic solution of fatty acid monoester, and keeping stand the mixture for 60 minutes; and 5, packaging the finished product by 200 liters per bucket for standby.