Studies of dynamic epigenomic changes during tumorigenesis using mice often require profiling epigenomes using a tiny quantity of tissue samples. Conventional epigenomic tests do not support such analysis due to the large amount of materials required by these assays. In this study, we developed an ultrasensitive microfluidics-based methylated DNA immunoprecipitation followed by next-generation sequencing (MeDIP-seq) technology for profiling methylomes using as little as 0.5 ng DNA (or ∼100 cells) with 1.5 h on-chip process for immunoprecipitation. This technology enabled us to examine genome-wide DNA methylation in a C3(1)/SV40 T-antigen transgenic mouse model during different stages of mammary cancer development. Using our data, we identified differentially methylated regions and their associated genes in different periods of cancer development. Our results showed that unique methylomic features were presented in various tumor developmental stages.