Abstract An important topic in systems biology is the reverse engineering of regulatory mechanisms through reconstruction of context-dependent gene networks. A major challenge is to identify the genes and the regulations specific to a condition or phenotype, given that regulatory processes are highly connected such that a specific response is typically accompanied by numerous collateral effects. In this study, we design a multi-layer approach that is able to reconstruct condition-specific genes and their regulation through an integrative analysis of large-scale information of gene expression, protein interaction and transcriptional regulation (transcription factor-target gene relationships). We establish the accuracy of our methodology against synthetic datasets, as well as a yeast dataset. We then extend the framework to the application of higher eukaryotic systems, including human breast cancer and Arabidopsis thaliana cold acclimation. Our study identified TACSTD2 (TROP2) as a target gene for human breast cancer and discovered its regulation by transcription factors CREB, as well as NFkB. We also predict KIF2C is a target gene for ER−/HER2− breast cancer and is positively regulated by E2F1. The predictions were further confirmed through experimental studies. Availability: The implementation and detailed protocol of the layer approach is available at http://www.egr.msu.edu/changroup/Protocols/Three-layer%20approach%20to%20reconstruct%20condition.html. Contact: krischan@egr.msu.edu Supplementary information: Supplementary data are available at Bioinformatics online.