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 20 performs pretreatment including a step of labeling a target site with a fluorescent dye to prepare a sample 20a. A fluorescence image analyzer 10 measures and analyzes the sample 20a. The fluorescent image analyzer 10 includes light sources 121 to 124 to irradiate light on the sample 20a, imaging part 154 to capture the fluorescent light given off from the sample 20a irradiated by light, and processing part 11 for processing the fluorescence image captured by the imaging part 154. The processing part 11 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 20a, and generates information used for determining whether the sample 20a is positive or negative based on the bright spots extracted for each of the plurality of cells.