The invention discloses a biological coal desulfurization device and a using method thereof. Rhodococcus erythropolis and thiobacillus ferrooxidans are mixed in a sulfuric acid environment at the pH value of 1-2, and inorganic sulfur in coal is removed by virtue of a biological reaction method. The biological coal desulfurization device mainly comprises a bacterium compound tank, a bacterium liquid metering pump, a pH value measuring instrument, a stirrer, a bio-reactor, a material delivery machine, a water delivery pump, a grinder, a liquid level transmitter, an acid and iron fetching tank, adrying machine, a washing machine, a filter, an air blower, a slurry pump, a PLC (Programmable Logic Controller) control system, an aeration coil pipe, a temperature transmitter, a sulfuric acid metering pump, a sulfuric acid storage tank, a rhodococcus erythropolis culture tank and a thiobacillus ferrooxidans culture tank. The operating steps and using method of the device are given. The desulfurization time is greatly shortened, the desulfurization efficiency is high, the coal slurry and coal-washing wastewater can be recycled, emission of three wastes is avoided, and the biological coal desulfurization device is suitable for industrialized treatment and production.