Disease surveillance testing for emerging zoonotic pathogens in wildlife is a key component in understanding the epidemiology of these agents and potential risk to human populations. Recent emergence of SARS-CoV-2 in humans, and subsequent detection of this virus in wildlife, highlights the need for developing new One Health surveillance strategies. We used lymph node exudate, a sample type that is routinely collected in hunter-harvested white-tailed deer (WTD, Odocoileus virginianus) for surveillance of chronic wasting disease, to assess anti-SARS-CoV-2 neutralizing antibodies. A total of 132 pairs of retropharyngeal lymph nodes collected from Nebraska WTD harvested in Nebraska, US, in 2019 (pre–SARS-CoV-2 pandemic) and 2021 (post–SARS-CoV-2 pandemic) were tested for SARS-CoV-2 with reverse transcription PCR. Thereafter, exudates obtained from these same lymph nodes were tested for SARS-CoV-2 neutralizing antibodies using a surrogate virus neutralization test. Neutralizing antibodies were detected in the exudates with high diagnostic specificity (100% at proposed cutoff of 40% inhibition). Application of this testing approach to samples collected for use in other disease surveillance activities may provide additional epidemiological data on SARS-CoV-2 exposure, and there is further potential to apply this sample type to detection of other pathogens of interest.