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Molybdenum disulfide nanosheets (MoS2NSs) show great promise in the field of optical biosensors due to its unique optical transparency and semiconducting properties. In this work, supramolecule calix[4]arene (pSC4) modified MoS2NSs (pSC4-MoS2NSs) was synthesized through a one-step preparation method. The strong p-π binding and electrostatic interactions between pSC4and MoS2NSs increased the stripping efficiency by preventing aggregation of nanosheets. Based on pSC4-MoS2NSs, a simple and feasible surface plasmon resonance (SPR) sensor was proposed. SPR refractive index change experiments were conducted to evaluate the sensor’s sensitivity performance, and the three main parameters of sensitivity were improved. Benefiting from the modification of pSC4, proteins were captured with higher surface coverage through the host-guest recognition, which is identical to optimization analysis experiment. According to a bovine serum albumin antibody immune experiment, pSC4-MoS2NSs based SPR sensors provided a new sensitive sensing interface with a 50.85% SPR signal increased compared that of MoS2NSs modification. Therefore, supramolecule modified MoS2NSs is promising for application to SPR immunosensing for the detection of cancer biomarkers.We reported a facile and versatile method for one-step preparing supramolecule modified MoS2nanosheets. Calix[4]arene, as stripping and protein adsorption reagent, was used to functionalize MoS2nanosheets with enhanced stripping efficiency, protein adsorption. The pSC4-MoS2NSs provided good optical properties for an enhanced sensitivity of the SPR sensor.