Mesoporous silica nanoparticles (MSN) were coated by pH-responsive polymer chitosan-poly (methacrylic acid) (CS-PMAA). This nano drug delivery system showed good application prospects and the polymer-coated microspheres were promising site-specific anticancer drug delivery carriers in biomedical field. A continuous detection of pH-responsive drug delivery system in cells in situ, utilizing MSN/CS-PMAA composite microspheres, was proposed. Two kinds of different cell lines, tumor cell line (Hela) and normal somatic cells (293T), were used to investigate the behaviours of the drug loaded system in the cells. Conclusions could be drawn from the fluorescent images obtained by confocal laser scanning microscopy (CLSM), modified drug-loaded microspheres (MSN/CS-PMAA) were ingested into cells more easily, the uptake of DOX@FITC-MSN/CS-PMAA by HeLa/293T cells were performed at pH 7.4/pH 6.8, DOX was released during the ingestion process, fluorescence intensity decreased with time because of efflux transport and photo-bleaching. Fluoresence detection by flow cytometry was performed as comparison. The continuous fluorescent observation in situ could be widely used in the pH-responsive releasing process of drug delivery system in the cells.