A plug-and-play surface plasmon resonance (SPR) dual-parameter optical fiber biosensor is reported, in which Au film was firstly coated on the fiber surface for exciting SPR and the end half of the Au film was modified with Au nanoparticles to generate double SPR resonance valleys. For simultaneous detecting of glucose and cholesterol concentrations, modified P-mercaptophenylboronic acid (PMBA) and β-cyclodextrin (β-CD) were subsequently coated on the surface of sensor probe. Due to the cis-diol structure of glucose, it can interact with PMBA, leading to a red shift of one SPR resonant valley, whose maximum wavelength shift is 11.228 nm in the range of 0–1.7 mM glucose concentration. On the same time, the cholesterol molecules can realize the host-guest combination with β-CD, leading to a red shift of another SPR resonant valley, and the maximum wavelength shift is 18.893 nm in the cholesterol concentration range of 0–300 nM. The detection limits of the sensor to glucose and cholesterol are 0.00078 mM and 0.012 nM, respectively. The enhances the practical value of the dual-parameter sensor. Both theory and experiment results verify the feasibility of the “plug-and-play” sensor to measure the dual biomass of glucose and cholesterol with ultra-low detection limit and good selectivity. The proposed method provides a huge research value for the optical fiber sensor in multi-parameter measurement. •A highly-sensitive and reflective optical fiber SPR biosensor was proposed for dual-parameter measurement.•Glucose and cholesterol concentrations could be simultaneously measured by one sensor.•Au nanoparticles were coated on the sensor to adjust the resonant wavelength and enhance the sensor sensitivity.•The cross-sensitivity between glucose and cholesterol could be neglected by selecting PMBA and β-CD as sensitive materials.•The sensor has an ultra-low detection limit and excellent selectivity for detecting low concentrations of glucose and cholesterol.