The invention relates to a handheld fluorescence detector which comprises a clamping groove, a first photoelectric switch, a second photoelectric switch, a light source, a light path system, and a photoelectric sensor, a signal amplification circuit, a peak holding circuit, a microprocessor and an analog-to-digital converter which are connected in order. The first photoelectric switch and the second photoelectric switch are mounted on the clamping groove and sequentially sense the reagent cards inserted into the clamping groove. The light path system comprises a first convex lens, a dichroic mirror, a second convex lens, an optical filter and a third convex lens; and light emitted by the light source sequentially penetrates through the first convex lens, is reflected to the second convex lens through the dichroic mirror, penetrates through the second convex lens and forms a light spot on a tested reagent card, and exciting light sequentially penetrates through the second convex lens, the dichroic mirror, the optical filter and the third convex lens to reach the photoelectric sensor. The detector is provided with a precise optical excitation and collection detection system, signals of the positions of the quality control line and the test line of the reagent card can be independently collected, the calculated amount is small, the speed is high, and the size of the detector can be smaller.