Calcium (Ca2+) is a pivotal and universal second messenger in cells. Changes in the intracellular concentration of Ca2+ are responsible for initiating, progressing, and completing various cellular processes, including fertilization. In mammals, fertilization triggers a series of Ca2+ oscillations inside the egg. The Ca2+ rise activates Ca2+/calmodulin-dependent protein kinase II (CaMKII), a serine/threonine kinase. CaMKII activity is required for cell cycle resumption or exit from the metaphase II (MII) stage of arrested eggs. In mammals, genetic experiments involving the knockout or down-regulation of CaMKII showed that female mice were sterile despite their eggs showing the ability to initiate regular Ca2+ oscillations. A series of well-structured studies unveiled that CaMKII activity corresponded with the early distinct Ca2+ increases within the first hour post-fertilization. Intriguingly, it has been revealed that CaMKII is sensitive to the frequency of Ca2+. Despite these findings, the complete profile, and alterations of CaMKII activity in oocytes remain unexplored. Many of the studies so far have been egg-lysate-based or dependent on in vitro assays. Hence, there is an evident lack of efficient tools to measure CaMKII's real-time activity. To study the CaMKII activity, we have developed a genetically encoded Förster resonance energy transfer (FRET)-based biosensors that provide a powerful approach for monitoring real-time endogenous CaMKII activity. We have characterized the responses of these CaMKII biosensors through various Ca2+ stimuli and have shown that the sensors are efficient, specific, and can monitor CaMKII activity for a long time. Additionally, we have elucidated the role of phosphatases, which are critical for regulating CaMKII activity.