Nowadays, the most common approaches in the prognosis, diagnosis, and treatment of diseases are along with undeniable limitations. Thus, the ever-increasing need for using biocompatible natural materials and novel practical modalities is required. Applying biomaterials, such as chitosan nanoparticles (CS NPs: FDA-approved long-chain polymer of N-acetyl-glucosamine and D-glucosamine for some pharmaceutical applications), can serve as an appropriate alternative to overcome these limitations. Recently, the biomedical applications of CS NPs have extensively been investigated. These NPs and their derivatives can not only prepare through different physical and chemical approaches but also modify with various molecules and bioactive materials. The potential properties of CS NPs, such as biocompatibility, biodegradability, serum stability, solubility, non-immunogenicity, anti-inflammatory properties, appropriate pharmacokinetics and pharmacodynamics, and so forth, have made them excellent candidates for biomedical applications. Therefore, CS NPs have efficiently applied for various biomedical applications, like regenerative medicine and tissue engineering, biosensors for the detection of microorganisms, and drug delivery systems (DDS) for the suppression of diseases. These NPs possess a high level of biosafety. In summary, CS NPs have the potential ability for biomedical and clinical applications, and it would be remarkably beneficial to develop new generations of CS-based material for the future of medicine.