Many studies have incorporated concept maps into digital games to enable learners to make connections between subject concepts in the game. However, most learners do not associate spontaneously with the thematic concepts in the game but need to be facilitated by effective scaffolding mechanisms to reconceptualize the learning process and content. Therefore, this study proposed a knowledge check-based concept map-guided gaming strategy for chemistry learning for secondary-level learners. Specifically, the knowledge check-based concept map-guided gaming strategy was employed as a learning strategy while playing digital games. The knowledge check mechanism was developed based on the notation of two-tier testing. Moreover, the learning environment was a self-developed chemical classification-based digital game, in which learners learned through concept mapping guided gaming tasks with factual and conceptual questions to confirm that they can actually learn content and conceptual understanding in the game. To understand the effects of the proposed strategy, a quasi-experimental design was adopted to measure compare two groups' learning achievement, problem-solving awareness, critical thinking tendency, and germane cognitive load of seventh-grade students using the knowledge check-based concept map-guided gaming strategy and the conventional concept map-guided gaming strategy. The results of the study showed that learners' learning achievement, problem-solving awareness, critical thinking tendency, and germane cognitive load in chemistry learning were effectively improved by the knowledge check-based concept map-guided gaming strategy. Furthermore, this study implies that teachers can adjust their teaching scaffolds and provide strategies according to learners' learning behaviors in digital game-based learning.