Chitosan–pDNA nanoparticles with various weight ratios (chitosan:pDNA 1:4–8:1) were characterized for particle size, zeta potential, morphology, and pDNA binding efficiency. For targeted gene delivery applications, nanoparticles were functionalized by coupling fluorescent dye and tyrosine kinase receptor B (TrkB) binding peptides on the particle surface. The targetability of the peptide-functionalized nanoparticles was demonstrated in TrkB positive murine transformed monocyte/macrophage cells (RAW 264). It was observed that weight ratio influenced DNA condensation and nanoparticle properties. An increase in the weight ratio decreased the average particle size, but increased the zeta potential. Cell culture studies showed that TrkB-peptide-functionalized nanoparticles bound to cells more effectively than nanoparticles functionalized with a control peptide. The length of the PEG spacer arm of the amine-to-sulfhydryl crosslinker used in the functionalization was found to positively correlate with the cellular attachment efficiency. This study suggests that the peptide-functionalization could be used to target chitosan–pDNA nanoparticles to specific cells.