Live microbial contamination poses high risks to cell and gene therapies, threatening manufacturing processes and patient safety. Rapid, sensitive detection of live microbes in complex environments, such as CAR-T cell cultures, remains an urgent need. Here, an innovative sample-to-result workflow is introduced using digital loop-mediated isothermal amplification (dLAMP), enhanced by Electrostatic Microfiltration (EM)-based enrichment, for rapid sterility testing. By rationally designing primers targeting 16S and 18S rRNA, dLAMP assay enables both universal detection (covering >80% of known species) and strain-specific identification of bacterial and fungal contaminants in CAR-T cell spent medium and final products, directly from microorganism lysates. Enhanced by EM-based enrichment of low-abundance live microbes, the workflow achieves unparalleled sensitivity and speed, detecting contamination levels as low as 1 CFU/mL in complex CAR-T cell cultures within 6 h. Compared to qPCR and 14-day compendial methods, the approach demonstrates superior accuracy and significantly faster turnaround times. This workflow holds transformative potential for real-time monitoring in cell therapy manufacturing and rapid safety assessments of CAR-T cell products prior to patient infusion. Beyond cell therapy, the method is broadly applicable to infectious disease diagnostics, biomanufacturing monitoring, food safety, and environmental surveillance.