Biotechnological production processes are sustainable approaches for the production of biobased components such as amino acids for food and feed industry. Scale‐up from ideal lab‐scale bioreactors to large‐scale processes is often accompanied by loss in productivity. This may be related to population heterogeneities of cells originating from isogenic cultures that arise due to dynamic non‐ideal conditions in the bioreactor. To better understand this phenomenon, deeper insights into single‐cell physiologies in bioprocesses are mandatory before scale‐up. Here, a triple reporter strain (3RP) was developed by chromosomally integrating the fluorescent proteins mEmerald, CyOFP1, and mTagBFP2 into the L‐phenylalanine producing Escherichia coli strain FUS4 (pF81kan) to allow monitoring of growth, oxygen availability, and general stress response of the single cells. Functionality of the 3RP was confirmed in well‐mixed lab‐scale fed‐batch processes with glycerol as carbon source in comparison to the strain without fluorescent proteins, leading to no difference in process performance. Fluorescence levels could successfully reflect the course of related process state variables, revealed population heterogeneities during the transition between different process phases and potentially subpopulations that exhibit superior process performance. Furthermore, indications were found for noise in gene expression as regulation strategy against environmental perturbation.