This paper reports a novel method for the statistical analysis of quantum dot (QD) cytotoxicity and cellular uptake based on single cell cycles, which is part of a series of works on the study of QD cytotoxicity using a microfluidic system (Lab Chip, 2012, 12, 3474–3480; 2013, 13, 1948–1954). The specially designed microfluidic system consisted of a polydimethylsiloxane (PDMS) microwell array for single-cell arrangement and microchannels for QD solution diffusion, enabling effective control of stable cell density and the interdistance between them, as well as maintaining a constant QD concentration with no disturbance of the fluids which can affect cellular uptake. We showed that the treatment of QDs had no influence on cell cycles. However, the QD cytotoxicity was found to be dependent on cellular uptake in various cell cycle phases, because the accumulation and dilution of QDs happened in single cell cycles. The rank of QD cytotoxicity was G2/M > S > G0/G1. Thus, this technology could serve as a new strategy to investigate otherwise inaccessible mechanisms governing nanoparticle cytotoxicity.