The anterior cruciate ligament (ACL) reconstruction mostly relies on the experience of surgeons. To improve the effectiveness and adaptability of the tension after ACL reconstruction in knee joint rehabilitation, this paper establishes a lateral force measurement model with relaxation characteristics and designs an on-line stiffness measurement system of ACL. In this paper, we selected 20 sheep knee joints as experimental material for the knee joint stability test before the ACL reconstruction operation, which were divided into two groups for a comparative test of single-bundle ACL reconstruction through the anterolateral approach. The first group of surgeons carried out intraoperative detection with routine procedures. The second group used ACL on-line stiffness measurement system for intraoperative detection. After that, the above two groups were tested for postoperative stability. The study results show that the tension accuracy is (− 2.3 ± 0.04)%, and the displacement error is (1.5 ± 1.8)%. The forward stability, internal rotation stability, and external rotation stability of the two groups were better than those before operation (P < 0.05). But the data of the group using the system were closer to the preoperative knee joint measurement index, and there was no significant difference between them (P > 0.05). The system established in this paper is expected to help clinicians judge the ACL reconstruction tension in the operation process and effectively improve the surgical effect.