Partial hydrolysis catalyzed by phospholipase A1 (Lecitase Ultra) in a solvent free system was firstly used to produce diacylglycerols (DAGs)-enriched soybean oil. In this study, five reaction parameters namely agitation speed (100–500rpm), reaction time (2–10h), water content (10–50wt% of oil mass), enzyme load (5–40U/g of oil mass), and reaction temperature (30–70°C) were investigated. The reaction was up-scaled to 1kg of soybean oil at 40°C of reaction temperature, with 300rpm of agitation speed, 40wt% of water content, 6h of reaction time and 22U/g of enzyme load. Purification by molecular distillation yielded 70% DAG-enriched oil with 42.64wt% of DAG. The composition of acylglycerols of soybean oil and the DAG-enriched soybean oil was analyzed and identified by high performance liquid chromatography (HPLC) and HPLC/electrospray ionization/mass spectrometer. The released fatty acid from the partial hydrolysis of soybean oil catalyzed by phospholipase A1 showed a higher saturated fatty acid content than that of the raw material. Compared to the lipase catalyzed process, this new phospholipase A1 catalyzed one showed the advantages of low amount production of byproduct, namely, monoacylglycerols.