Abstract: Combined with template molecular pre-positioning, surface imprinting method, and post-modification strategy, a molecularly imprinted surface plasmon resonance (SPR) sensor was constructed on a gold substrate and used for detection of β-lactoglobulin (BLG) in camel milk powder. Firstly, the template molecule BLG was fixed by mercapto-terminated polyacrylic acid (PAA-SH) on the gold substrate which was modified by polydopamine in advance. Then imprinting was performed using dopamine as functional monomer and crosslinking agent. Finally, partially hydrolyzed poly (2-methyl-2-oxazoline) (PMOXA-EI) with anti-protein adsorption function was grafted on the non-imprinted part, and the BLG molecularly imprinted SPR sensor was prepared after eluting the template. The changes of sensor’s surface hydrophilicity/hydrophobicity were studied by water contact angle (WCA) and the characterization of created sensor was performed by Fourier transform infrared spectrometer (FT-IR), atomic force microscopy (AFM), and variable angle spectroscopy ellipsometry (VASE), respectively. Results show that the sensor performs a good linear relationship in the range of 0.21—10 μg/mL for BLG, and the detection of limit is 0.12 μg/mL. The sensor could detect the BLG in camel milk powder quickly and sensitively with a recovery rate between 100.2% and 100.7%.