Mechanical properties of biological tissues are significant biomarkers for diagnosing various diseases. Assessing the viscoelastic properties of multi-layer tissues has remained challenging for a long time. Some shear wave models have been proposed to estimate thin-layer tissues' viscoelasticity recently. However, the potential applications of these models are highly restricted since few biological tissues are single-layered. Here we proposed a multi-layer model for layer-specific viscoelasticity estimation of biological tissues. Integrating the theoretical model and ultrasonic micro-elastography imaging system, the viscoelasticity of both layers was assessed. Dual-layer phantoms and ex vivo porcine eyes were used to verify the proposed model. Results obtained from the mechanical test and shear wave rheological model using bulk phantoms were provided as validation criteria. The representative phantom had two layers with elastic moduli of 1.6 ± 0.2 kPa and 18.3 ± 1.1 kPa, and viscosity moduli of 0.56 ± 0.16 Pa·s and 2.11 ± 0.28 Pa·s, respectively. The estimated moduli using the proposed model were 1.3 ± 0.2 kPa and 16.20 ± 1.8 kPa, and 0.80 ± 0.31 Pa·s and 1.87 ± 0.67 Pa·s, more consistent with the criteria (one-tailed t-test, p < 0.1). By contrast, other methods, including the group velocity method and single-layer Rayleigh-Lamb model, generate significant errors in their estimates. For the ex vivo porcine eye, the estimated viscoelasticity was 23.2 ± 8.3 kPa and 1.0 ± 0.4 Pa·s in the retina, and 158.0 ± 17.6 kPa and 1.2 ± 0.4 Pa·s in the sclera. This study demonstrated the potential of the proposed method to significantly improve accuracy and expand clinical applications of shear wave elastography.