Ensuring the authenticity of meat products is crucial for food safety. In this study, we proposed a method employing a combination of time-resolved fluorescent microsphere-based lateral flow assay and polymerase chain reaction (TRFM-LFA-PCR) for quantitatively detecting beef content to assess the authenticity of beef products, without the need of knowing the types of adulterants. The PCR amplicons (45 min) were detected by TRFM-LFA and the results can be observed within 20 min. This approach had demonstrated the ability to detect beef at concentrations as low as 0.005%. Furthermore, a calibration curve was established between the T/C ratio and the logarithmic value of beef content (0.1%-100%), with an R2 value of 0.99. The TRFM-LFA-PCR method was applied to detect adulterated samples, with recoveries between 82.91% and 116.29% and relative standard deviation ranging from 2.90% to 13.72%. Consequently, TRFM-LFA-PCR shows great potential for detection of beef adulteration without the need of knowing the types of adulterants.