Pioneering electron energy loss spectroscopy (EELS) measurements of α-Bi2O3 are performed on three samples obtained through different synthesis methods. Experimental low-loss and core-loss EELS spectra are acquired. By combining them with detailed structural characterization and Density Functional Theory (DFT) simulations, we are able to detect and evaluate the presence of oxygen vacancies in the samples. This type of information has not been accessed previously from EELS data in bismuth oxide, because high-resolution EELS spectra or how vacancies reflect in Bi2O3 spectra were unreported. This novel measurement is further validated through comparison with photoluminescence data. Therefore, the technique has the ability to probe oxygen vacancies in Bi2O3 at an unprecedented resolution, which might allow solving material science and technological issues related to this material.