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• Electric field intensity of multilayer of GNPs coupled with gold film was studied. • The RI sensitivity of the double-layer gold nanospheres sensor was 15747 nm/RIU. • The RI sensitivity of the double-layer gold nanorods sensor was 25642 nm/RIU. • The LOD of double-layer gold nanospheres biosensor is 10 ng/mL. • The LOD of double-layer gold nanorods biosensor is 4.6 ng/mL. The main challenge for all biosensors is how to measure the biological samples at very low concentrations. In this paper, a surface plasmon resonance (SPR) fiber biosensor using multi-layer gold nanoparticles (Au NPs)/Au film coupling to enhance sensitivity was proposed. The coupling effect between such longitudinally aligned multilayer nanoparticles increases the intensity of the local electromagnetic field and the depth of propagation to the outside. We used the finite element method to analyze the local enhancement effect of multi-layer Au nanospheres and Au nanorods, and the fabricated double-layer Au NPs (Au nanospheres and Au nanorods)/Au film coupling enhancement sensors. These two sensors' refractive index sensitivities are 15,747 nm/RIU and 25,642 nm/RIU, respectively. By changing biomolecules immobilized on the sensor's surface, the highly sensitive double-layer Au nanoparticle/Au film fiber sensor enables high-precision measurement of different biomarkers at low concentration. For detection of human IgG, the detection limit of gold nanosphere sensor is 10 ng/mL and gold nanorod sensor is 4.6 ng/mL. [ABSTRACT FROM AUTHOR]