One of the main obstacles of Photodynamic Therapy (PDT) to damage and destroy abnormal cells is that most photosensitizers (Ps) have a highly hydrophobic nature with a tendency to aggregate in aqueous solutions and the non-specificity towards target cells. Nanotechnology proposes new tactics for the development of monomeric Ps nanotransporters and active targeting mole-cules with the use of biodegradable polymeric nanoparticles to improve the specificity towards target cells. The goal of this work was to optimize the synthesis of chitosan polymeric nanoparticles conjugated with protoporphyrin IX and vitamin B9 (CNPs-PpIX-B9) by the ionic gelation method from the established protocol previously carried out by our laboratory with 1.74 times fold of efficiency. They were characterized by ultraviolet-visible and infrared spectroscopy and transmission electron microscopy. The optimal conditions for CNPs synthesis was found at pH 5.11. The nanoconjugate shapes were more homogeneous and the average size resulted in 19.92 nm ± 7.52 nm. CNPs-PpIX-B9 were stable after the filter sterilization method and highly thermostable.