The oncogenic mutation heterogeneity of the cancer cell population has been proven to be essential for predicting both drug-response and drug-resistance of targeted therapies, such as tyrosine kinase inhibitors. It is necessary to accurately evaluate the mutation heterogeneity, oncogenic mutation and resistant mutation profiling at a single cell level. However, there are two major hurdles in the process. First, majority of the cells in tumor tissue are non-cancer cells, which cause background noise. Second, the work load and cost of next generation sequencing on dozens of single cells are prohibitive. To address both these issues, we developed a microfluidic chip for profiling of dozens of selected cells. With the help of a novel tri-states valve structure, which performs precise controlling of the cell/reagent movement, as well as active mixing of different reagents, trapping/identification/lysis and in situ MDA amplification was achieved at a single cell level on the same chip. Using a proof-of-concept assay mimicking EGFR targeting drug Gefitinib treatment of lung cancer cells, the new method was validated as capable of not only detecting the existence of multiple mutations, but also providing complete information of the mutation scenario at the single cell level by using cost-effective Sanger's sequencing.