Circular dichroism (CD) spectra contain information about absolute configurations and conformations of chiral compounds. However, extracting this information from CD spectra in solution is challenging, because the spectra exhibit only the averaged CD values of all different conformers. CD spectroscopy of jet-cooled molecules can provide conformation-specific CD spectra, but its application to biomolecules has been limited due to the difficulty of their production in the gas phase. Here, we obtained the first CD spectra of chiral molecular ions produced by electrospray ionization (ESI) using cold ion CD spectroscopy. Protonated l- or d-phenylalanine ions produced by ESI were stored in a cold quadrupole ion trap and irradiated by multiple laser pulses with left or right circular polarization. The CD spectra exhibited well-resolved CD bands of two conformers, whose signs were opposite to each other. This study will broaden the scope of conformation-resolved CD spectroscopy to large molecular ions without size limitations.