The conjugation of tRNA with chitosan nanoparticles of different sizes 15,100 and 200kDa was investigated in aqueous solution using multiple spectroscopic methods and atomic force microscopy (AFM). Structural analysis showed that chitosan binds tRNA via G-C and A-U base pairs as well as backbone PO2 group, through electrostatic, hydrophilic and H-bonding contacts with overall binding constants of KCh-15-tRNA=4.1 (±0.60)×103M−1, KCh-100-tRNA=5.7 (±0.8)×103M−1 and KCh-200-tRNA=1.2 (±0.3)×104M−1. As chitosan size increases more stable polymer-tRNA conjugate is formed. AFM images showed major tRNA aggregation and particle formation occurred as chitosan concentration increased. Even though chitosan induced major biopolymer structural changes, tRNA remains in A-family structure.