Purpose The aim of this study was to determine the anatomic, operative and biological factors that inluenced graft healing after single-bundle anterior cruciate ligament (ACL) reconstruction. Methods One hundred fourteen consecutive patients who underwent anatomic single-bundle ACL reconstruction with quadrupled hamstring tendon autografts between 2016 and 2019 were retrospectively analyzed. Ninety-four patients met the inclusion criteria with minimum follow-up of 12 months. Patients were evaluated with multiple clinical measurements, including International Knee Documentation Committee Subjective Knee Form (IKDC-SKF), Lyshom Scores, and Marx activity scale. To evaluate graft healing, the signal-to-noise quotient (SNQ) was measured at intra-articular graft and intratunnel integration were evaluated on magnetic resonance imaging (MRI) at one year after surgery. Potential factors afecting graft healing, including age, sex, body mass index, time from injury to surgery, posterior tibial slope, lateral femoral condyle ratio, notch width index, meniscal injury, remnant preservation, tunnel aperture locations, graft size, graft bending angle, graft/remaining notch volume ratio were evaluated for their association with graft SNQ value by stepwise regression analysis. Results A total of 94 patients were evaluated with mean follow-up 28.5 ± 9 months. Univariate regression analysis showed that posterior tibial slope, notch width index, remnant preserving procedure, high femoral tunnel, anterior tibial tunnel, graft bending angle, and graft/remaining notch volume ratio signiicantly associated with graft SNQ values. Multivariate regression analysis showed that lateral tibial slope, remnant preservation, and graft/remaining notch volume ratio were independent factors correlated with graft SNQ values. Also, the graft SNQ values was weakly correlated with femoral tunnel integration and Marx activity scale at one year. There was no correlation between graft SNQ values and IKDC-SKF and Lysholm scores. There was no correlation between graft SNQ values and International Knee Documentation Committee and Lysholm scores. Conclusions Tibial slope, remnant preservation and graft/remaining notch volume ratio were signiicant independent associated factors of graft SNQ value at one year. The graft SNQ values were also weakly correlated with femoral tunnel integration and the Marx activity scale. These factors should be taken into account for ensuring the ideal graft healing and for the return to sport decision-making. Level of evidence Level IV.