An embodiment of the present disclosure provides a multi-reflection silicon-based liquid immersion micro-channel measurement device and measurement method capable of improving measurement sensitivity by completely separating, through multi-reflection, first reflective light reflected by a sample detection layer and a second reflective light by a prism-buffer solution interface and by allowing the light to enter multiple times through the multi-reflection. The multi-reflection silicon-based liquid immersion micro-channel measurement device according to the embodiment of the present disclosure includes a micro-channel structure including a support, and one or more micro-channels formed on the support and each having a sample detection layer with a fixed bioadhesive material for detecting a sample, a sample injection unit configured to inject a buffer solution containing the sample into the micro-channel, a prism unit including a prism, and a reflection structure formed by coating a bottom surface of the prism with a mirror reflection material, the polarized light generating unit configured to generate polarized light, and the polarized light detecting unit configured to detect a polarization change of reflected light.