Abstract: The disclosure provides a microfluidic detection chip, a preparation method thereof, a fixing device and a centrifugal detection device. The detection chip comprises overlapped three layers. The upper layer of the chip includes a sample loading area and vents; the lower layer of the chip includes a waste liquid tank area in which a slope structure or a groove is disposed; the intermediate layer of the chip is a double-sided adhesive layer on which sample flow channels are divided by an adhesive area and an adhesive-free area. The self-driving and short-time centrifugation combined microfluidic detection chip technology designed by the present disclosure can solve inherent problems of traditional paper substrates, further improve sample utilization, detection speed and detection sensitivity in immunoassay, and the preparation and assembly of the chip are simple, has low requirement for the detection device, and can be conveniently applied to the clinical detection.