Investigating how AI-enhanced teaching influences pre-service teachers’ instructional application competence in digital-intelligent literacy is of substantial value for optimizing teacher education curricula, improving instructional management in teacher preparation programs, and enhancing the quality of future teachers’ digital-intelligent development. Taking the course AI-Enhanced Instructional Design Innovation and Application as an example, this study employed a self-developed questionnaire to examine changes in pre-service teachers’ digital application competence and practical AI application competence before and after course participation. A total of 299 valid responses were collected from students enrolled at a normal university in Yunnan Province, China. Drawing on the digital application competence dimension in the Teacher Digital Literacy framework and the practical application dimension in the AI Literacy Framework for Primary and Secondary School Teachers in Guangdong Province (Trial), this study conducted a pre-post comparative analysis of pre-service teachers’ instructional application competence in digital-intelligent literacy. The findings indicate that participation in the course significantly improved students’ competence, particularly in digital instructional design, digital instructional implementation, intelligent resource development, and intelligent instructional implementation. However, relatively limited gains were observed in digital academic assessment, intelligent instructional evaluation, and collaborative education.