AbstractThe SARS‐CoV‐2 non‐structural protein 14 (nsp14), known as exoribonuclease is encoded from the large polyprotein of viral genome and is a major constituent of the transcription replication complex (TRC) machinery of the viral RNA synthesis. This protein is highly conserved among the coronaviruses and is a potential target for the development of a therapeutic drug. Here, we report the SARS‐CoV‐2 nsp14 expression, show its structural characterization, and ss‐RNA exonuclease activity through vibrational and electronic spectroscopies. The deconvolution of amide‐I band in the FTIR spectrum of the protein revealed a composition of 35 % α‐helix and 25 % β‐sheets. The binding between protein and RNA is evidenced from the spectral changes in the amide‐I region of the nsp14, showing protein conformational changes during the binding process. A value of 20.60±3.81 mol L⁻¹ of the binding constant (K_D) is obtained for nsp14/RNA complex. The findings reported here can motivate further studies to develop structural models for better understanding the mechanism of exonuclease enzymes for correcting the viral genome and can help in the development of drugs against SARS‐CoV‐2.