The invention relates to an in vitro diagnostic reagent and a preparation method thereof and in particular relates to a rapid detection kit for drug susceptibility of mycoplasma bovis and a preparation method of the rapid detection kit. The invention provides the rapid detection kit for the drug susceptibility of the mycoplasma bovis; the kit comprises a mycoplasma culture solution, a drug susceptibility plate and an antibiotic drug. The mycoplasma culture solution is prepared from the following components according to the content: 10 to 15g of PPLO (Pleuropneumonia-like Organism) powder, 2 to4g of oligopeptide, 100 to 500mg of morroniside, 2 to 6g of yeast powder, 1 to 3g of glucose, 1.5 to 4.5g of sodium pyruvate, 80 to 120mL of an MEM culture medium, 2mL of a 1 percent (w/v) phenol redsolution, 100 to 300mL of horse serum, 0.3 million units of penicillin and 700 to 800mL of ddH2O. The culture solution provided by the invention can be used for rapidly promoting the growth of the mycoplasma bovis and detection can be finished within 20 to 24h; the rapid detection kit has stable performance and strong specificity.