G-quadruplexes are common structural motifs in aptamers. UNA or unlocked nucleic acid is the latest nucleic acid modification. We have attempted to evaluate the impact of UNA modification on the structure and stability of G-quadruplex oligonucleotides for application in aptamer design. We show using CD spectroscopy that UNA modifications can cause structural transitions in some cases although they retain the inherent G-quadruplex signature. From UV melting studies we showed a position dependent effect of UNA modifications such that quadruplexes with UNA modified loops are further stabilized whereas UNA modifications in stem of the G-quadruplex significantly destabilize the structure. The impact of UNA modification on different nucleobases is also investigated. From the analysis of UV melting results, thermodynamic profile was computed and it was concluded that all the sequences are stable at 37 °C. Finally, a greater serum stability of the modified oligonucleotides in comparison with unmodified ones is also demonstrated. Overall, the position dependent effect of single UNA substitutions was observed and analysed.