Provided is a fluorescence image acquisition apparatus for acquiring fluorescence images and phase images using optical signals that are modulated at the same frequency and that have different time delays. The fluorescence image acquisition apparatus may include a light source configured to generate, at different time delays, a plurality of optical signals that are modulated at the same frequency, an illuminator configured to control paths of the plurality of modulated optical signals so that the plurality of modulated optical signals are illuminated onto a sample including a plurality of fluorescent materials, a photodetector configured to detect a plurality of fluorescence signals that are emitted from the plurality of fluorescent materials, respectively, and a controller configured to acquire a plurality of fluorescence images and a plurality of phase images from the plurality of detected fluorescence signals.