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The drug binding to serum protein (SP) is an area of intense research in evaluating drug candidates. Thus, composite material based on chitosan (CS), and amoxicillin tri hydrate (AMOT) were designed in order to evaluate the release of AMOT@CS NPs and it’s binding with BSA under physiological condition. The inclusion of 2% v/v T80 in the CS NPs enabled in vitro release of AMOT, at 298 K and pH 7.4 up to 68.03%. This study also was undertaken to compare and explore the binding of potential chemotherapeutic antibacterial drug Amoxicillin trihydrate (AMOT) and AMOT@Chitosan nanoparticles (AMOT@CS NPs) with a model protein bovine serum albumin (BSA) by fluorescence spectroscopy, synchronous fluorescence spectroscopy, Ultraviolet–visible (UV–vis) absorption, and CD spectroscopic techniques. Experimental results indicated that AMOT and AMOT@CS NPs could bind with BSA and quench the fluorescence of BSA via static mechanism. The interactions among BSA with AMOT, AMOT/T80 and AMOT@CS NPs were evidenced by substantial changes in the BSA secondary structure, as revealed by circular dichroism. The findings of thermodynamic parameters revealed that the binding reaction in both systems was exothermic and spontaneous, enthalpically driven and that the hydrogen bonding and Vander Waals forces played a vital role to achieve optimal interaction between AMOT/ AMOT@CS NPs and BSA.