In this study, we utilized proteins to control the assembly of split DNAzyme to establish protein-controlled split DNAzymes (Pc SD), with the aim of enhancing its catalytic activity. To achieve this, simultaneous recognition of protein by affinity ligands at both ends of split DNAzyme fragments induced an increased local concentration of each split fragment, leading to reassembly of the split catalytic core with a rigid conformation and higher affinity to its cofactor. As a result, under protein control, Pc SD exhibits unexpected cleavage efficiency compared to free split DNAzyme. To further explore the catalytic features, we then systematically positioned split sites within the catalytic core of three popular DNAzyme-based Pc SDs, thus revealing the important nucleic acids that influence Pc SDs activity. Based on the excellent analytical performance of Pc SD for streptavidin (with a LOD of 0.1 pM in buffer),we equipped Pc SD with antibodies as rapid diagnostic tools for inpatient care (AFP as biomarker) with a minimized workflow (with a LOD of 2 pM in 5% human serum). The results of this study offer fundamental insights into external factors for boosting DNAzyme catalysis and will be promising for applications that utilize split DNAzymes.