A fluorescence image analyzer, analyzing method, and pretreatment evaluation method capable of determining with high accuracy whether a sample is positive or negative are provided. A pretreatment part performs pretreatment including a step of labeling a target site with a fluorescent dye to prepare a sample. A fluorescence image analyzer measures and analyzes the sample. The fluorescent image analyzer includes light sources to irradiate light on the sample, imaging part to capture the fluorescent light given off from the sample irradiated by light, and processing part for processing the fluorescence image captured by the imaging part. The processing part extracts the bright spot of fluorescence generated from the fluorescent dye that labels the target site from the fluorescence image for each of a plurality of cells included in the sample, and generates information used for determining whether the sample is positive or negative based on the bright spots extracted for each of the plurality of cells.