The invention relates to a microfluidic chip based on optical detection and a preparation method thereof. A microfluidic chip based on optical detection includes an upper substrate layer, a lower substrate layer and an interlayer. The interlayer is positioned between the two substrate layers, the interlayer is provided with a sample channel and an injection hole, and the sample channel comprises a groove structure penetrating through the interlayer and substrate surfaces. The preparation method comprises the following steps: 1, preparing the groove structure and the injection hole in the interlayer, wherein the groove structure penetrates through the interlayer; and 2, bonding the interlayer with groove structure and the two substrates together, so that side wall of the groove and the substrate surfaces constitute the sample passage. The invention has the beneficial effects that the surface of the microfluidic chip sample channel is smooth, so no thermal mismatch exists; and arrangement of the injection hole leads to turbulence within the sample channel, so as to improve the detection accuracy. Therefore, the invention has the characteristics of simple preparation process, high stability and high detection sensitivity.