Abstract To address the challenges associated with sample injection into the air hole of photonic crystal fiber (PCF) 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 °C −1 , and the refractive index sensitivity is 1666.2 nm RIU −1 . This structure solves the difficulty of filling and coupling PCFs. Furthermore, it introduces a novel research methodology for the design and assembly of high-performance biosensors based on PCFs.