Microbial testing slip is a new detection tool which is convenient to carry and free of media preparation. Since the key technique of the testing slip is cold water gelling agent, a new cold water gelling agent (HKG)’s water retention capacity, pH value, gels strength, source utilization by microorganisms and inhibition effect were tested in this assay. Furthermore, detection effect and specificity of HKG total plate count bacterial testing slip of food samples were compared with pour-plate method in order to evaluate its feasibility of gelling agent in microbial testing slip. HKG showed no antibacterial effect to 16 kinds of microorganisms, also the features of neutral pH, gels strength of 340.31 g/cm2 ± 48.71 g/cm2, good water retention capacity, HKG can not support the growth of microbe or be degrading by microorganism in short time (5 d). HKG testing slip had the same sensitivity (P = 0.438 > 0.05) as pour plate method in detection of 5 strains, the colony count of various test strains (P = 0.442 > 0.05) and the colony count of food samples (P = 0.718 > 0.05) of HKG testing slip had no significant difference in statistics compared with pour plate method. As the gelling agent, HKG shows good performance in bacterial testing slip, and can also improve the detection efficiency.