The utility model relates to a device for preparing anaerobic culture fluid, which relates to a cell culture device. The device aims at solving the problems that the anaerobic environment of an existing culture dish is insufficient, which is not beneficial to the cell growth, and anaerobic culture fluid which meets the requirements can not be prepared. A nitrogen steel cylinder, a nitrogen and carbon dioxide compound steel cylinder or a hydrogen gas and carbon dioxide compound steel cylinder is connected with a first air passage pipe, wherein a second air passage pipe is communicated with a left side port on a three-mouth flask, an extractor is arranged on a sucking port on the three-mouth flask, a heater is arranged under the three-mouth flask, a third air passage pipe is connected with a vacuum air pump, an air distributor is arranged on a charging port on the third air passage pipe, an anaerobic charging pipeline is connected with the charging port and is communicated with a culture fluid bottle, a flexible pipe is connected with the extractor and is communicated with the culture fluid battle. The device can fully form an anaerobic environment in the spaces in the three-mouth flask and the culture fluid bottle, and is beneficial to the cell growth, thereby the anaerobic culture fluid meeting the requirements can be prepared.