Imaging-based spatial-omics advances biomedical discoveries with subcellular resolution and high sensitivity, but accurately identifying signal spots from diverse images remains challenging. We develop U-FISH, a deep learning method that enhances images for consistent spot detection across various spatial-omics data. We establish a comprehensive FISH image dataset from seven spatial-omics methods. Benchmark analysis shows U-FISH has superior accuracy and generalizability compared to existing methods and can effectively decode 3D FISH data. U-FISH is the first spot detection software integrated with large language models, as demonstrated in AI-assisted diagnostics. Our study provides a valuable tool for spatial-omics analysis and diagnostics.