The invention belongs to the technical field of microculture and in particular relates to mycobacteria enrichment fluid and a preparation method and application thereof. The mycobacteria enrichment fluid comprises broth, brain heart infusion broth, biotin, glycerin, sodium polyanethol sulfonate, Tween 80, bovine serum albumin, oleic acid and distilled water. The preparation method comprises the following steps: weighing raw materials, mixing the raw materials, regulating the pH value, sterilizing, weighing the biotin and bovine serum albumin, filtering, degerming and mixing the sterile raw materials. According to the mycobacteria enrichment fluid disclosed by the invention, the culture time of mycobacteria is short, the growth conditions of the mycobacteria can be simply detected, the detection period is short, the positive rate is high, and the mycobacteria in a clinical blood specimen can be effectively separated and cultured.