Cell behaviors are known to be regulated by the cellular microenvironment. Traditional cell-population based analysis methods need to separate cells from their extracellular matrix (ECM) and cannot resolve the heterogeneity of cell behaviors. Herein, an in situ single-cell analysis method based on rolling circle amplification was exploited to image gene expression in single cells for investigating the effect of ECM stiffness on cell growth. This method enables the simultaneous quantifying of the cell phenotype and gene expression at the single-cell level, which can help in understanding the underlying molecular mechanism of cell growth. It is found that ECM stiffness could affect cell growth via regulating the expression level of the cytoskeleton-assembly associated genes PFN1 and CFL1 and their co-expression pattern. Therefore, this single-cell analysis platform may facilitate us to tap into the study of “single-cell phenotypes” and elucidate the disease association of ECMs.