Background This study investigated the anticoagulant properties of sulfated chitosan derived from the internal bone of the spineless cuttlefish . Chitosan, a biopolymer, is used in various biomedical applications including anticoagulation. Sulfation of chitosan enhances its biological activity, making it a potential therapeutic agent. This study explored the efficacy of sulfated chitosan in preventing blood clot formation to provide a novel anticoagulant alternative. Objectives This study aimed to synthesize and characterize the anticoagulant properties of sulfated chitosan extracted from the internal bone of the spineless cuttlefish using Fourier Transform Infrared Spectroscopy (FTIR), Field Emission Scanning Electron Microscopy (FESEM), and X-Ray Diffraction (XRD) and evaluate the anticoagulant properties of sulfated chitosan extracted from the internal bone of spineless cuttlefish . Materials and methods Chitin and chitosan were extracted from the cuttlebone of a specimen of , and sulfated chitosan was synthesized by sulfation of chitosan. Sulfated chitosan was subsequently used to evaluate its anticoagulant properties using tests such as activated partial thromboplastin time (APTT) and prothrombin time (PT). Characteristic investigations were conducted, including FTIR, FESEM, and XRD analyses. Results The results of this study suggested the possibility of using internal bone as an unconventional source of natural anticoagulant that can be combined with biomedical applications. Anticoagulant activity measured using APTT and PT showed that sulfated chitosan was a strong anticoagulant. Conclusion We examined the anticoagulant activity of extract using thrombin and activated partial thromboplastin times. Our results demonstrated the heparin-like anticoagulant action of the extracted sulfated chitosan, suggesting that it may be a great alternative anticoagulant treatment.