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Steroidal molecularly imprinted polymers (MIPs) and non-imprinted polymer (NIP) were prepared by precipitation polymerization method using cholesterol, deoxycholic acid, and β-sitosterol as the virtual templates, acrylic acid (AA) as the functional monomer, azobisisobutyronitrile (AIBN) as the initiator, and ethylene glycol dimethacrylate (EGDMA) as the cross-linking agent. The morphology and structure of the polymers were characterized using scanning electron microscopy (SEM), X-ray spectroscopy (XRD), Fourier-transform infrared spectroscopy (FT-IR), and Brunauer-Emmett-Teller (BET) specific surface area analysis. The adsorption performance for steroidal substances was also investigated. The results showed that the steroidal MIPs were uniformly-sized and well-dispersed spherical nanoparticles with porous surface. Adsorption performance results showed that MIPs had significantly stronger adsorption capacities for 5α-cholestane compared to NIP. Among the three MIPs, deoxycholic acid and β-sitosterol MIPs demonstrated stronger adsorption capacities for 5α-cholestane than that of cholesterol. The adsorption kinetics studies showed that the adsorption process of MIPs for 5α-cholestane was in accordance with the pseudo-second-order kinetic model, which was mainly controlled by chemisorption. The isothermal adsorption of both MIPs and NIP conformed to the Langmuir isothermal adsorption model and the Scatchard model, indicating that MIPs exhibited specific selective adsorption for 5α-cholestane and that the adsorption process was monolayer adsorption, with a maximum adsorption capacity of 0.735 mg/g. The study suggests that MIPs prepared with cholesterol, deoxycholic acid, and β-sitosterol as virtual templates have high molecular recognition and selectivity for 5α-cholestane.