Background: The purpose of this study was to quantify and compare the biomechanical characteristics of a new locking loop stitch (LLS), developed utilizing the concepts of both running locking stitch and needleless stitch, to the traditional Krackow stitch. Methods: The Krackow stitch with No.2 braided suture and the LLS with 1.3-mm augmented polyblend suture tape were compared biomechanically. The LLS was performed with single strand locking loops and wrapping suture around the tendon, resulting in half the needle penetrations through the graft compared to the Krackow stitch. Twenty bovine extensor tendons were divided randomly into two groups. The tendons were prepared to match equal thickness and cross-sectional area. Each suture-tendon was stitched and preloaded to 5 N for 60 seconds, cyclically loaded to 20 N, 40 N, and 60 N for 10 cycles each, and then loaded to failure. The deformation of the suture-tendon construct, stiffness, yield load, and ultimate load were measured. Results: The LLS had significantly less deformation of the suture-tendon construct at 100 N, 200 N, 300 N, and at ultimate load compared to the Krackow stitch (Krackow stitch and LLS at 100 N: 1.3 ± 0.1 mm and 1.0 ± 0.2 mm, p < 0.001; 200 N: 3.0 ± 0.3 mm and 1.9 ± 0.2 mm, p < 0.001; 300 N: 5.1 ± 0.6 mm and 2.9 ± 0.4 mm, p < 0.001; ultimate load: 12.8 ± 2.8 mm and 5.0 ± 1.2 mm, p < 0.001).The LLS had significantly greater stiffness (Krackow stitch and LLS: 97.5 ± 6.9 N/mm and 117.2 ± 13.9 N/mm, p < 0.001) and yield load (Krackow stitch and LLS: 66.2 ± 15.9 N and 237.9 ± 93.6 N, p < 0.001) compared to the Krackow stitch. There was no significant difference in ultimate load (Krackow stitch: 450.2 ± 49.4 N; LLS: 472.6 ± 59.8 N; p = 0.290). Conclusions The LLS had significantly smaller deformation of the suture-tendon construct compared to the Krackow stitch. The LLS may be a viable surgical alternative to the Krackow stitch for graft fixation when secure fixation is necessary.