The morphology of tRNA was studied upon conjugation with testosterone and its aliphatic and aromatic dimers, using multiple spectroscopic methods, transmission electron microscopy (TEM) and molecular modeling. Structural analysis showed that testosterone binds tRNA through A62, A64, C60, C61, C63, G51, U50 and U59 bases. The binding affinity was testosterone dimer-aromatic > testosterone dimer-aliphatic > testosterone. The steroid loading efficacy was 35–45%. Transmission electron microscopy showed major changes in tRNA morphology upon testosterone interaction with an increase in the diameter of the tRNA aggregate, indicating encapsulation of testosterone by tRNA.