We described a simple technique for the fabrication of arc-edge-channel monolithic valves (AMVs) as the core component in a microfluidic device. AMVs were compatible for multiplexed control (based on binary patterns), large-scale integration, flowing control, microparticle manipulation and single cell viability detection. Furthermore, in combination with an allele specific locked nucleic acid (LNA)-based quantitative PCR (AS-LNA-qPCR) assay developed in this study, the microfluidic device was used to isolate single cells from well-established lung cancer cell lines and clinical samples for analysis of common target mutations at single cell level. The results showed that mutation heterogeneity exists in both cell lines and clinical samples, indicating the importance of single cell analysis. The detection of both EGFR (T790 M) single mutation and EGFR (L858R and T790 M) double mutations in cancer cells isolated from one pleural effusion sample with original L858R single mutation revealed the association of drug-resistance status with new T790 M mutation. These results demonstrated the feasibility of a low-cost and simple method of AMVs fabrication for liquid control and microparticle manipulation, and the practicality of a low-cost and efficient alternative for clinical gene mutation detection and heterogeneity study at the single cell level by combining AMVs with AS-LNA-qPCR.