To address the challenges associated with sample injection into the air hole of photonic crystal fiber and collimation, in this paper, we assemble a single-mode photonic crystal single-mode fiber structure sensor chip based on the Mach-Zehnder interference principle using microfluidic chip processing technology. The sensing principle is analyzed mathematically and the sensing characteristics are verified theoretically and experimentally. The temperature sensitivity of the sensor is −1.3325 nm/℃, and the refractive index sensitivity is 1666.2 nm/RIU. This structure solves the difficulty of filling and coupling photonic crystal fibers. Furthermore, it introduces a novel research methodology for the design and assembly of high-performance biosensors based on photonic crystal fibers.