Single-nucleotide polymorphisms (SNPs) are closely related to human diseases and individual drug responses, and the accurate detection of SNPs is crucial to both clinical diagnosis and development of personalized medicine. Among various SNPs detection methods, ligase detection reaction (LDR) has shown great potential due to its low detection limit and excellent specificity. However, frequent involvement of expensive labels increases the experimental cost and compromises the assay efficiency, and the requirement of careful predesigned probes limits it to only known SNPs assays. In this research, we develop a controllable mismatched ligation for bioluminescence screening of both known and unknown mutations. Especially, the ligation specificity of E. coli ligase is tunable under different experimental conditions. The mismatches locating on the 3'-side of the nick cannot be ligated efficiently by E. coli ligase, whereas all mismatches locating on the 5'-side of the nick can be ligated efficiently by E. coli ligase. We design a 3'-discriminating probe (3'-probe) for the discrimination of known mutation and introduce a T7 Endo I for the detection of unknown mutation. With the integration of bioluminescence monitoring of ligation byproduct adenosine 5'-monophosphate (AMP), both known and unknown SNPs can be easily detected without the involvement of any expensive labels and labor-intensive separation. This method is simple, homogeneous, label-free, and cost-effective and may provide a valuable complement to current sequencing technologies for disease diagnostics, personalized medicine, and biomedical research.