An objective-type dark-field illumination device for a microfluidic channel is provided and includes an optical stop having a pair of symmetric curved slits used to adjust the optical path and inner numerical aperture of a dark-field light source generated by the device. The dark-field illumination can focus on a smaller spot to illuminate a sample in the microfluidic channel by matching a pin-hole combined with a transmitter objective lens. The optical path and smaller spot is advantageous to solve the problem of a traditional dark-field illumination that may generate background light noise scattered from inner walls of the microfluidic channel to lower the image contrast. Therefore, the signal or image resolution of capturing the scattered light and/or emitted fluorescent light emitted from the sample in the microfluidic channel can be enhanced. Meanwhile, the device can simultaneously excite and detect multiple fluorescent samples with different excited wavelengths in the microfluidic channel.