Pursuit of an ultrafast and highly efficient method to fabricate homogeneous carbon quantum dots (CQDs) with high quantum yield is a persistent objective for photoluminescence (PL) mechanism and bioimaging. Here, the CQDs were fabricated through ablation of low-cost carbon cloth by ultrafast and highly efficient dual-beam pulsed laser ablation. Unlike previous reports, a single laser beam is divided into double beams by a beam splitter in this work for shortening laser ablation time and improving ablation yield. More importantly, the obtained CQDs are more homogeneous than those obtained by single-beam pulsed laser ablation, and the corresponding quantum yield of emission is as high as 35.4%. The homogeneous CQDs show a stable and excitation-independent PL emission, which is attributed to the electron transition from the π∗ state to the surface state rather than from the π∗ state to the π state based on our experimental evidence. Furthermore, the CQDs have a good stability and excellent antijamming performance, which are well suitable for cell bioimaging. Therefore, a ‘dual-beam pulsed ablation’ technique proposed as an ultrafast method is used to fabricate homogeneous CQDs with high quantum yield for the study of PL emission mechanism and cell bioimaging.