A detection device, including a light-emitting component, a light-detecting component, at least one reflective optical film element, and a control unit, is provided. The light-emitting component is used for providing an excitation beam, wherein a part of the excitation beam whose dominant wavelength falls within an excitation wavelength band generates a fluorescence beam after passing through a test specimen. The light-detecting component is used for receiving a part of the fluorescence beam whose dominant wavelength falls within a detection wavelength band. The control unit is coupled to the at least one reflective optical film element. The control unit controls the at least one reflective optical film element to filter out a part of a wavelength band of an incident beam. The incident beam is at least one of the excitation beam and the fluorescence beam. A detection method of the detection device is also provided.