The invention discloses a borrelia BSK storage liquid culture medium and a single colony separating and purifying method and application thereof. The borrelia BSK storage liquid culture medium comprises CMRL-1066, bovine serum albumin V, new peptone, hydroxyethyl piperazine ethanesulfonic acid, sodium citrate, glucose, yeast powder, sodium bicarbonate, sodium pyruvate, acetyl glucosamine, rabbit serum, sodium hydroxide, and the like. The single colony separating and purifying method comprises the following steps of: preparing the BSK storage liquid culture medium, preparing sepharose gel, sterilizing at high pressure, gradually melting, uniformly mixing with the BSK storage liquid culture medium in proportion, and pouring to prepare a lower solid culture medium; mixing a borrelia burgdorferi bacterium suspension diluted in a gradient way and the BSK storage liquid culture medium, pouring into an upper culture medium so that a macroscopic single colony appears; raising the single colony by using a toothpick, inspecting by using a dark-field microscope to observe a representative burgdorferi bacterium strain. The method disclosed by the invention has the advantages of easiness and fastness for operation and clarity in result. According to the invention, the representative burgdorferi bacterium strain is uniform in genetic background without heterogeneity, suitable for subsequent scientific research and used as a standard strain.