The commercialization of industrial genetically modified microorganisms (GMMs) has highlighted their impact on public health and the environment. Rapid and effective monitoring methods detecting live GMMs are essential to enhance current safety management protocols. This study aims to develop a novel cell-direct quantitative polymerase chain reaction (qPCR) method targeting two antibiotic-resistant genes,KmRandnptII, conferring resistance against kanamycin and neomycin, along with propidium monoazide, to precisely detect viableEscherichia coli. TheE. colisingle-copy taxon-specific gene of D-1-deoxyxylulose 5-phosphate synthase (dxs) was used as the internal control. The qPCR assays demonstrated good performance, with dual-plex primer/probe combinations exhibiting specificity, absence of matrix effects, linear dynamic ranges with acceptable amplification efficiencies, and repeatability for DNA, cells, and PMA-treated cells targetingKmR/dxsandnptII/dxs. Following the PMA-qPCR assays, the viable cell counts forKmR-resistant andnptII-resistantE. colistrains exhibited a bias% of 24.09% and 0.49%, respectively, which were within the acceptable limit of ±25%, as specified by the European Network of GMO Laboratories. This method successfully established detection limits of 69 and 67 viable genetically modifiedE. colicells targetingKmRandnptII, respectively. This provides a feasible monitoring approach as an alternative to DNA processing techniques to detect viable GMMs.