This paper developed a ready-to-use quality control sample which can be directly used without secondary culture for the rapid detection of Escherichia coli in meat and meat products by matrix assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF-MS). The sample was evaluated for homogeneity and stability. The results showed that the survival rate of the quality control sample prepared using the selected cryoprotectant was above 90%. The culture count result of the quality control sample (F = 0.567) was less than the critical value, suggesting good uniformity. The quality control sample was stable for 14 days at 37 and 25 ℃, and the resuscitation rates were 101.5% and 99.6% at −20 and 4 ℃ for 28 days, respectively. After 28 days of storage at 37 and −20 ℃, the results of MALDI-TOF-MS were consistent, indicating that the developed sample had good stability and met the requirements of quality control samples.