Microscopy imaging of living cells is becoming a pivotal, noninvasive, and highly specific tool in metabolic engineering to visualize molecular dynamics in industrial microorganisms. This review describes the different microscopy methods, from fluorescence to super resolution, with application in microbial bioengineering. Firstly, the role and importance of microscopy imaging is analyzed in the context of strain design. Then, the advantages and disadvantages of different microscopy technologies are discussed, including confocal laser scanning microscopy (CLSM), spatial light interference microscopy (SLIM), and super-resolution microscopy, followed by their applications in synthetic biology. Finally, the future perspectives of live-cell imaging and their potential to transform microbial systems are analyzed. This review provides theoretical guidance and highlights the importance of microscopy in understanding and engineering microbial metabolism.