Naringin (Nar), a natural flavanone glycoside, has been shown to possess a variety of biological activities, including anti-inflammatory, anti-apoptotic and bone formation, etc. In this study, Nar was oxidized by sodium periodate and the oxidized naringin (ONar) was used as a novel biological crosslinking agent. And ONar-fixed porcine decellularized Achilles tendon (DAT) was developed to substitute anterior cruciate ligament (ACL) for researching a novel ACL replacement materials. The ONar with a 24 h oxidation time had appropriate aldehyde group content, almost no cytotoxicity, and a good crosslinking effect. The critical characteristics and cytocompatibility of ONar-fixed DAT were also investigated. The results demonstrated that 1% ONar-fixed DAT exhibited good resistance to enzymatic degradation and thermal stability as well as suitable mechanical strength. Moreover, 1% ONar-fixed specimens exhibited excellent L929 fibroblasts-cytocompatibility and MC3T3-E1-cytocompatibility. They also promoted the secretion of hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) from fibroblasts and bone morphogenetic protein-2 (BMP-2) from osteoblasts. And they also showed the good anti-inflammatory properties in vivo experiments. Our data provided an experimental basis for ONar as a new cross-linking reagent in chemical modification of DAT and ONar-fixed DAT for ACL repair.