Background Saliva and blood, being biological materials with a high potential for infectious transmission in dental environments, pose significant risks to dental professionals, assistants, and patients alike. Therefore, practitioners must adopt stringent security measures to ensure patient care, considering all parties as potential carriers of microorganisms capable of causing infectious diseases. Currently, various methods of disinfection and sterilization are employed to maintain the aseptic chain effectively. Having reliable methods for detecting substances in liquids, particularly body fluids, is crucial and highly convenient. Luminol, a chemiluminescent agent widely used in forensic science for detecting minute traces of blood that are invisible to the naked eye, presents itself as a valuable tool. Blood, a major bodily fluid often present in instruments following dental procedures, underscores the importance of its detection. Hence, in this study, luminol was utilized to detect blood traces in dental instruments following dental treatment, both before and after sterilization or disinfection. Objective Blood and saliva splashes, together with highly contagious aerosols, are always a part of dental procedures. The objective of the current study is to detect traces of blood stains on face shields, surgical instruments, and endodontic files using luminol before and after sterilization. Materials and methods Sample size calculation was done with G*Power software (Version 3.1.9.4, Düsseldorf, Germany), and a total of 30 instruments were selected for the study. In the present study, a total of 30 items were collected and utilized, including 14 instruments used after implant placement, 12 endodontic files employed after root canal treatment, and four face shields utilized during these procedures. Meanwhile, a freshly prepared luminol solution was applied to these instruments, and they were viewed in a dark environment both before and after sterilization procedures. Luminescence generated by luminol was observed in the instruments, indicative of the presence of blood not visible to the naked eye. Statistical analysis for both groups was done with IBM SPSS Statistics for Windows, Version 16.0 (Released 2007; SPSS Inc., Chicago, IL, USA). Intragroup comparison was done using the Friedman test, and intergroup comparison was done using the Wilcoxon signed-rank test. Results Blood stains and chemiluminescence were visualized in two out of 10 endodontic files (one #15 K-file and #20 K-files) and two out of four face shields. The intragroup comparison was done using the Friedman test, and it was found to be statistically significant (p < 0.05). Intergroup comparison was done using the Wilcoxon signed-rank test and was found to be statistically insignificant (p > 0.05). Conclusion Following sterilization and disinfection, there were no visual blood stains or chemiluminescence. Therefore, luminol was found to be effective in detecting blood stains in endodontic files, surgical instruments, and face shields, as well as in validating the sterilization and disinfection processes. Hence, sterilization in dentistry stands as a critical measure to guarantee patient safety, halt the dissemination of infections, and uphold exemplary clinical care standards.