Diagnosing neurosyphilis in clinical settings poses significant challenges due to the absence of highly efficient diagnostic criteria. Our objective was to enhance the existing diagnostic criteria and assess their sensitivity and specificity for identifying neurosyphilis in HIV-negative patients. We conducted a retrospective review of patient records from a cross-sectional study carried out between December 2019 and May 2023. This study was designed to explore clinical practices in the diagnosis and treatment of neurosyphilis. We proposed and validated a new set of diagnostic criteria across 782 cases, comparing three different NS criteria for diagnostic efficiency. Among the 782 patients analyzed, 410 were diagnosed with neurosyphilis, while 372 were not. The newly developed NS criteria demonstrated a sensitivity of 99.5% and a specificity of 94.6%, with the added benefit of being user-friendly. In subgroups categorized by neurological symptoms and syphilis stages, the new NS criteria also exhibited high sensitivity and specificity. Statistical analyses confirmed that the novel NS criteria performed comparably well to the expanded NS criteria. The novel NS criteria for HIV-negative patients exhibited high sensitivity and specificity, proving to be both effective and convenient for physicians to implement.