Effects of suture preparation on graft contamination remain unknown in anterior cruciate ligament reconstruction (ACLR). This study aimed to evaluate the incidence of allograft contamination at different time points of graft preparation and investigate differences in contamination between different sites of the allografts. Fourteen hamstring tendon (HT), 9 quadriceps tendon (QT), and 9 bone-patellar tendon-bone (BTB) allografts were harvested, sterilised, and stored following routine procedures. Graft suture preparation was performed with baseball stitching for soft tissue and bone drilling for bone plug. The time was recorded simultaneously. The graft was kept moist in a standard operating room environment for 30min after the initiation of preparation. The specimens were obtained from the middle and both ends of each graft for culture at three different time points: pre-suturing, post-suturing, and 30min after the initiation of preparation. A total of 192 specimens were transferred to the microbiology laboratory for culture, identification, and semi-quantitative assessment. Culture results were classified as negative, poor, and abundant based on the extent of growth. Contamination level was recorded as low or high corresponding to culture results of poor or abundant. The duration of suture preparation was 348, 301, and 246s for HT, QT, and BTB (P = 0.090). The specimens had a positive culture rate of 41/192 (21.4%), of which 21 were from the ends and 20 from the middle. More positive samples with abundant bacterial growth were detected from the ends than from the middles post-suturing (7/8 vs. 1/7, P = 0.010) and at 30min (6/11 vs. 0/11, P = 0.012). The total graft contamination rate was significantly higher at 30min (19/32, 59.4%) than pre-suturing (4/32, 15.6%) and post-suturing (9/32, 28.1%) (P < 0.001). The contamination rate with abundant bacterial growth was higher post-suturing (7/32, 21.9%) than pre-suturing (0%). No statistically significant differences were found among the three types of allografts. The contamination rate increases significantly at 30min compared with pre-suturing and post-suturing. Suture preparation may have introduced the high-level contamination, to which the ends of the graft were more prone than the middle. Therefore, routine prophylactic decontamination after suture preparation should be considered, especially for the ends of the grafts.