Background The effect of effective length on the biomechanical properties of the graft is regarded as an essential variable influencing the posterior cruciate ligament reconstruction. However, the effect has not been fully studied. The purpose was to compare the effects of different effective graft lengths (35 mm, 55 mm, 65 mm) on the time-zero biomechanical properties of the graft in posterior cruciate ligament (PCL) reconstruction.Methods Bovine digital flexor tendons and porcine tibias were used to establish in-vitro PCL reconstruction models. Tensile strength testing was performed at 3 different effective lengths of the graft: short (35 mm, n = 10, group 1), medium (55 mm, n = 10, group 2), and long (65 mm, n = 10, group 3). A computer software (Trapezium X; Shimadzu) was used to record the load-elongation curve, ultimate load (N), the elongation of the graft during the test (mm), tensile stiffness (N/mm), and energy absorption (J). The failure pattern was evaluated by visual observation.Results All the samples failed because the grafts slipped out from the bones, except two grafts ruptured in group 1. The tensile stiffness and ultimate load in group 1 were significantly higher than those in group 2 and group 3 (tensile stiffness, 50.49 ± 11.43 N/mm in group 1 vs 31.20 ± 10.44 N/mm in group 2[P < 0.001] and 19.18 ± 6.18 N/mm in group 3 [P < 0.001]; ultimate load, 452.40 ± 54.52 N in group 1 vs 338.50 ± 26.79 N in group 2 [P < 0.001]and 268.70 ± 28.30 N in group 3 [P < 0.001]). There were significant differences between group 1 and group 3 in energy absorption(9.61 ± 3.25 J vs 5.22 ± 2.43 J, P = 0.002). At 50 N and 100 N of applied load, no statistically significant differences were detected on the elongation of the grafts (P > 0.05). The elongation of the short grafts under 150 N and 200 N of applied load was significantly less than that of the long grafts (150 N, 1.77 ± 0.83 mm in group 1 vs 4.14 ± 1.75 mm in group 3, P = 0.047; 200 N, 2.62 ± 1.10 mm in group 1 vs 7.06 ± 3.20 mm in group 3, P = 0.006).Conclusions This study demonstrated the time-zero biomechanical properties of the graft with short effective length were superior to those of the graft with medium and long effective lengths in PCL reconstruction.