This experiment was conducted to study on sample particle size, wash times of undigested residues, and preservation time of treated dialysis bag affecting the determination of dry matter digestibility (DMD) and enzymatic hydrolysate gross energy (EHGE) of corn and soybean meal using an in vitro digestion method based on simulative digestion system. It would provide a reference to develop a method for measuring the EHGE of feed using simulative digestion system. A single factor completely randomized design with 11 treatments was adapted. Each treatment contained 5 replicates with 1 digestion tube per replicate. The treatments 1-3, 4-7 and 8-11 were used to determine the effects of sample particle size, wash times of undigested residues, and preservation time on DMD and EHGE of corn and soybean meal, respectively. The results showed as follows: 1) sample particle size significantly affected the DMD and EHGE of corn and soybean meal (P<0.05). When particle size was decreased from 40 mesh to 100 mesh, the DMD and EHGE of corn were increased. The changes of DMD and EHGE of corn were both more obvious in particle size ranged from 40 mesh to 60 mesh. However, when particle size was decreased from 40 mesh to 100 mesh, the DMD and EHGE of soybean meal with 60 mesh were higher than those of the samples with 40 mesh and 100 mesh. And the difference of DMD of soybean meal with 40, 60 and 100 mesh was not more than 0.70%. 2) The wash times of undigested residues significantly affected the DMD and EHGE of corn and soybean meal (P<0.05). The DMD and EHGE of corn and soybean meal became stable when increasing wash times of undigested residues. After washing undigested residues for above 6 times, the variations of DMD and EHGE of corn and soybean meal both were in the permitted range of systematic error of simulative digestion system. 3) The preservation time of treated dialysis bag also significantly affected the DMD and EHGE of corn and soybean meal (P<0.05). However, in the 4 treatments, the differences of the DMD and EHGE of corn and soybean meal were not more than 0.94% and 0.37 MJ/kg, respectively, which were in the permitted range of systematic error of simulative digestion system. In conclusion, 60 mesh sample particle size and 6 times for washing undigested residues are the suitable condition in determination of EHGE of feed using the in vitro digestion method based on simulative digestion system. Preservation of treated dialysis bag for 90 d and at 4 ℃ don’t obviously affect the DMD and EHGE of corn and soybean meal.