Differential gene expression under particular conditions is tightly controlled at the transcriptional and post-transcriptional level. Transcription factors (TFs), key regulators at the transcriptional level, constitute a large and diverse group of double-strand DNA binding proteins which bind close to a target gene to activate or repress its transcription. Large-scale profiling of active transcription factors is essential to assist investigation into complicated gene regulatory networks as well as to help global pathway mapping. Here, a reproducible and reliable high-throughput mouse oligonucleotide array-based platform which can simultaneously explore the activities of nearly 200 different transcription factors is presented. The array comprises 240 synthetic probes which contain TF binding sequences based on 226 PSSMs provided by the TRANSFAC® database. The binding specificity between TF and its binding sequence was validated using antibody-based individual TF assay for AP1, HNF1, HNF4, NF1, NFκB, PPARγ and STAT1. Then purified NFκB was used to test the sensitivity and quantitative ability of the microarray. It could detect as little as 0.5 nmol/L of NFκB protein and maintain linear increase from 0.5 nmol/L to 20 nmol/L. Reproducibility was also explored using nuclear extracts from mouse macrophage RAW264.7 cells. The correlative coefficient between two independent experiments was 0.99 when using the identical nuclear protein. The reliability of the platform was then validated using two well studied cell models including LPS-stimulated MAPK pathway and IFNγ-stimulated JAK/STAT pathway. And finally, the TF profiling microarray was applied into the study of mouse preadipocyte 3T3-L1 differentiation. In addition to the well studied C/EBP family and PPAR family, two unknown transcription factors, SRF and NFAT, were also found changed in the process of adipocyte differentiation. Totally, a reliable microarray platform to profile the mouse transcription factor activities was successfully constructed.