Phosphorylation of histone H2AX (γH2AX) is a sensitive marker of activated DNA damage involving the formation of double-strand breaks. A novel quantum dot-labelled immunoassay was developed for profiling the genetic toxicity of cigarette smoke based on the quantitative detection of γH2AX expression in A549 cells. After stimulation, cells were fixed and permeabilised in the wells of 96-well plates for the measurement of γH2AX in whole cells, which eliminated the need of preparing cell lysates. A high-affinity mouse anti-γH2AX antibody and a normalisation antibody that recognised the total H2AX regardless of phosphorylation status were used for detecting the γ-H2AX and total H2AX in cells separately. Then two specific quantum dots-labelled secondary antibodies were added and combined specifically with the primary antibodies. The quantum dots with different excitation were used as sensitive markers for the existence of analytes with higher laser efficiency, larger stokes shift and excellent photostability. The fluorescence of detecting γH2AX is normalised to that of the total H2AX in each well for the correction of well-to-well variations. Due to the unique size-dependent optical properties of QDs, the fluorescence readout signal of this method was 5-fold higher than that of the traditional immunofluorescence assay which uses organic dye labelled-secondary antibody. A dose-dependent effect of cigarette smoke exposure on γH2AX expression was investigated and the contributions of cigarette smoke condensate and vapour phase components to the genetic toxicity of cigarette smoke were also discussed by comparing the γH2AX levels in stimulated cells.