Digital game-based learning is a student-centered learning environment that influences learning success and learning motivation. Whereas, a poorly designed instructional game will impair learning. Through extensive knowledge construction and high engagement, collaborative concept mapping strategies have the potential to address above problems. Simultaneously, the first principles of instruction could further enhance the effectiveness of collaborative concept mapping. Therefore, a collaborative knowledge organizer-based digital game utilizing the first principles of instruction approach was proposed for Chinese eighth graders in biology courses. This study adopted a quasi-experimental design with three groups of 92 students: a collaborative knowledge organizer-based digital game (CKO-DG) group (N = 33), a knowledge organizer-based digital game (KO-DG) group (N = 31), and a collaborative digital game (CDG) group (N = 28). The findings demonstrated that the CKO-DG students achieved the best biological achievement and motivation. However, the KO-DG students lacked peer support, resulting in a higher cognitive load; the CDG students lacked a proper strategy to organize their knowledge, resulting in low learning outcomes.