Vibrational circular dichroism (VCD) spectroscopy has a unique specificity to chirality and is highly sensitive to the conformational equilibria of chiral molecules. On the other hand, the matrix-isolation (MI) technique allows substantial control over sample compositions, such as the sample(s)/matrix ratio and the ratio among different samples, and yields spectra with very narrow bandwidths. We combined VCD spectroscopy with the MI technique to record MI-VCD and MI-vibrational absorption spectra of 3-butyn-2-ol at different MI temperatures, which allowed us to investigate the conformational distributions of its monomeric and binary species. Good mirror-imaged MI-VCD spectra of opposite enantiomers were achieved. The related conformational searches were performed for the monomer and the binary aggregate and their vibrational absorption and VCD spectra were simulated. The well-resolved experimental MI-VCD bands provide the essential mean to assign the associated vibrational absorption spectral features correctly to a particular conformation in case of closely spaced bands. By varying the matrix temperature, we show that one can follow the self-aggregation process of 3-butyn-2-ol and confidently correlate the MI-VCD spectral features with those obtained for a 0.1 M CCl4 solution and as a neat liquid at room temperature. Comparison of the aforementioned experimental VCD spectra shows conclusively that there is a substantial contribution from the 3-butyn-2-ol aggregate even at 0.1 M concentration. This spectroscopic combination will be powerful for studying self-aggregation of chiral molecules, and chirality transfer from a chiral molecule to an interacting achiral molecule and in electron donor–acceptor chiral complexes.