The existing screening methods for therapeutic targets of active ingredients in traditional Chinese medicine (TCM) have problems of long detection time and high instrument cost. This article proposes a new target screening method based on virtual screening and fiber surface plasmon resonance (SPR) sensing technology, which has the characteristics of flexibility, speed, and low cost. It also reveals the target mechanism of the active ingredient isoquercitrin in the treatment of insulin resistance (IR). The binding energies of isoquercitrin with target proteins PDPK1, INSR, and PTPN1 were calculated using computer virtual methods to be -8.9, -8.9, -8.8 kcal/mol, indicating strong binding activity with isoquercitrin and predicted as three key targets. Then a fiber optic SPR biosensor functionalized with isoquercitrin molecules was constructed to detect the binding affinity between isoquercitrin and the key targets. The experimental results showed that the binding affinities of isoquercitrin to the targets PDPK1, INSR, and PTPN1 were 1.45, 1.14, and 13.21, respectively, indicating that PTPN1 is the main target of isoquercitrin in the treatment of IR. The proposed sensor has a sensitivity of 0.699 nm/(μg/ml), LOD of 0.515μg/ml, and the experimental detection time of this method is as low as 45 minutes, without the need for large and expensive optical demodulation equipment, and the device volume is 5.50 dm