The current temperature-compensated fiber-optic surface plasmon resonance (SPR) biosensors are mainly open-ended outside the sensing structure, and there is a lack of temperature compensation schemes in fiber-optic microfluidic chips. In this paper, we proposed a temperature-compensated optical fiber SPR microfluidic sensor based on micro-nano 3D printing. Through the optical fiber micro-machining technology, the two sensing areas were designed on both sides of the same sensing fiber. The wavelength division multiplexing technology was used to collect the sensing light signals of the two sensing areas at the same time. The specific measurement of berberine and the detection of ambient temperature in the optical fiber SPR biological microfluidic channel were realized, and the temperature compensation matrix relationship was constructed, and then the temperature compensation was realized when measuring berberine biomolecules. Experiments have shown that the temperature sensitivity of the optical fiber SPR microfluidic sensor was 2.18 nm/°C, the sensitivity of the detection of berberine was 0.2646 nm/(µg/ml), the detection limit (LOD) was 0.38 µg/ml, and in a mixed solution showed an excellent specific detection impact.