The traditional finite element method-based fluorescence molecular tomography (FMT)/ X-ray computed tomography (XCT) imaging reconstruction suffers from complicated mesh generation and dual-modality image data fusion, which limits the application of in vivo imaging. To solve this problem, a novel standardized imaging space reconstruction (SISR) method for the quantitative determination of fluorescent probe distributions inside small animals was developed. In conjunction with a standardized dual-modality image data fusion technology, and novel reconstruction strategy based on Laplace regularization and L1-fused Lasso method, the in vivo distribution can be calculated rapidly and accurately, which enables standardized and algorithm-driven data process. We demonstrated the method's feasibility through numerical simulations and quantitatively monitored in vivo programmed death ligand 1 (PD-L1) expression in mouse tumor xenografts, and the results demonstrate that our proposed SISR can increase data throughput and reproducibility, which helps to realize the dynamically and accurately in vivo imaging.