Introduction: Pulse Oximetry (PO) is widely used for the noninvasive detection of hypoxemia, particularly in the perioperative setting. Research regarding the perioperative assessment of nursing knowledge of PO continues to evolve. Prior evaluations have identified relative knowledge deficits among nurses regarding physiologic principles, data interpretation, and clinical application. Such deficits need identification and targeted education to prevent perioperative patient mismanagement. Aim: To assess nursing knowledge of PO in perioperative and critical care settings of a single center in the United States and to analyse identified knowledge deficits. Materials and Methods: A cross-sectional questionnaire study was conducted in Post Anaesthesia Care Unit (PACU) and Medical Respiratory ICU (MRICU) among 150 participants. The validated questionnaire on surveying PO principle consisted of 21 true/false and 3 open-ended questions. Univariate analysis of nursing Total Knowledge Score (TKS), years of clinical experience, and current practice settings was performed. Significance was set at p<0.05. Results: A total of 51 questionnaires were completed, with a 34% response rate. The mean TKS for the true/false questionnaire was 16.4±2.0. There was no significant difference in TKS between PACU and MRICU nurses (16.5±1.7 vs. 16.2±2.5, respectively, p=0.27). Ninety-eight percent of all nurses scored >50% correct while 13.7% scored >90% (p=0.19 and p=0.74, respectively). The lowest percentages of correct responses (<50%) were seen in questions 3 and 16, which pertained to physiologic principles of PO. There was no significant difference in scoring percentiles between MRICU and PACU nurses, nor was there significant difference in TKS based on clinical experience. Conclusion: The mean TKS of nurses in the study was higher than the mean TKS of nurses in reference studies. There was no significant difference in TKS between PACU and MRICU nurses. Furthermore, clinical experience did not correlate with TKS scores. The authors believe analysing these knowledge deficits may offer educational opportunities in initial and continuing nursing education as well as routine postgraduate assessments.