The invention belongs to the technical field of fermentation and in particular discloses a process for preparing and extracting L-glutamine. The process comprises the following steps: preparing L-glutamine feed liquid, performing ultrafiltration treatment, removing mycoprotein and other macromolecular substances, performing activated carbon decolorization, strongly acidic cation exchange, vacuum concentration, isoelectric point cooling crystallization, centrifugation and the like on ultrafiltrate so as to obtain a crude product; re-dissolving the crude product, and sequentially performing weakly acidic and weakly alkaline ion exchange resin impurity removal, vacuum concentration, isoelectric point cooling crystallization, centrifugation and drying, thereby obtaining the refined L-glutamine. According to the process in the invention, the fermentation efficiency of the L-glutamine is improved, the finished product has high purity and total yield, the process is simple and convenient in production technology, the usage amount of the exchange resin is obviously decreased, and the amount of acid and alkali is greatly reduced, so that wastewater discharge is reduced, and environmental protection is facilitated.