The additive manufacturing (AM) process often results in non-uniform microstructure and different mechanical properties in sequential layers, impacting the overall performance of the AM-ed component. However, it is extremely challenging to evaluate the local stress-strain behavior of each individual layer, owing to the limited size of the AM-ed layered structure. To this end, a framework for characterizing and predicting the mechanical evolution of AM-ed multiphase alloys by combing nanoindentation and microstructure-based finite element method (FEM) was proposed. The sample used in this study was superduplex stainless steel (SDSS) manufactured by wire arc additive manufacturing (WAAM), and the microstructure varied from layer to layer. Firstly, the mechanical properties of the two constituent phases in each layer, including elastic modulus and hardness, were obtained by nanoindentation, and the indentation size effect (ISE) was also evaluated. The yield strength and hardening exponent of each phase were subsequently estimated by reverse analysis method, and therefore the constitutive behaviors of the individual phase, which served as input parameters for FEM, were acquired. By aid of real microstructure-based FEM under uniaxial tension, the overall stress-strain behaviors of each layer and the distributions of the stress and strain during the deformation process were investigated. This work provides a new avenue for the characterization of the multiphase alloys in AM industry, beneficial to the understanding of the mechanical evolution in AM-ed materials.