Cancer cells predominantly utilize aerobic glycolysis for energy production, preferentially converting glucose (Glu) to pyruvate (PA) and subsequently to lactate (LA). This metabolic reprogramming results in extracellular LA accumulation, acidifying the tumor microenvironment (TME) and facilitating tumor invasion and metastasis. The dynamics of Glu, PA, and LA are pivotal to tumor initiation and progression. This review comprehensively discussed therapeutic strategies targeting these key metabolites and systematically evaluates electrochemical and fluorescence-based techniques for their dynamic monitoring. We highlight the critical role of these monitoring approaches in advancing early cancer diagnosis, enabling personalized treatment, and accelerating anticancer drug development.