The shear characteristics of interlayer shear weakness zones (ISWZs) closely related to their material composition and structures are critical to the engineering stability of rock mass. To investigate ISWZs’ complex shear characteristics involving multiple factors, this study first proposes a novel remolded specimen preparation method involving the soil-rock interface, particle size distribution, the thickness of interlayer soil, water content, etc. Then, the sensitivity analysis involving the roughness of the soil-rock interface, the content of big particles, the thickness of interlayer soil, and normal stress based on the orthogonal design and significance test is studied. The results show that each factor's sensitivity for shear strength is in the order of normal stress > the thickness of interlayer soil > the content of big particles > the roughness of the soil-rock interface. Among them, the normal stress and the thickness of interlayer soil significantly affect shear strength, which is the primary factor. At the same time, the content of big particles and the roughness of the soil-rock interface are secondary factors. The influence of interlayer soil thickness and roughness of the soil-rock interface on the shear strength of ISWZs is determined by their relative relationship. When the fluctuation height of the soil-rock interface is generally greater than the thickness of interlayer soil, the shear strength is jointly controlled by the soil-rock interface and the interlayer soil. As the thickness of the interlayer increases, the effects of the soil-rock interface gradually weaken, and the interlayer soil mainly determines the shear strength of the ISWZ. The relevant research results are significant for the quantitative analysis and prediction of the shear strength of ISWZs.