The early diagnosis of diseases related to phosphate metabolism is very important for preventing, in most cases, severe kidney failure and cardiovascular events that have an increased risk of death. In this work, we present biofunctionalized quantum dots (b-QDs) prepared with chitosan and its chemically modified derivatives to detect and capture phosphate in water media. Chitosan (CHI), N,N,N-trimethylchitosan (TMC) and carboxymethylchitosan (CMC) were used as ligands for producing colloidal CdS nanocrystals in aqueous systems. UV-Visible spectroscopy (UV-Vis), photoluminescence spectroscopy (PL), fluorescence microscopy, Raman spectroscopy, Fourier transform infrared spectroscopy (FTIR), zeta potential analysis (ZP), and transmission electron microscopy (TEM) were used to characterize the colloidal QDs. Additionally, in vitro assays based on the b-QDs were designed and used to evaluate phosphate ion adsorption. The results clearly indicated that chitosan and chitosan derivatives were effective in nucleating and stabilizing CdS QDs in aqueous colloidal suspensions with an average nanocrystal size ranging from 2.2 to 3.6 nm. The photoluminescent behavior of the CdS bioconjugates was highly dependent on the chemical functionality introduced in the chitosan-derivative polymer. Moreover, the presence of different chemical groups in chitosan significantly affected the capacity of the bioconjugates to bind phosphates. Finally, a “pseudo in vivo” assay was designed using an artificial biomembrane based on phospholipid bilayers to preliminarily assess the possibility of using the bioconjugates to biolabel cell membranes. Therefore, a new class of fluorescent biosorbent nano-hybrids was developed using chemically functionalized chitosan conjugated to quantum dots for potential applications in biochemical analysis and nanomedicine.