Detecting EBV DNA load in nasopharyngeal (NP) brushing samples for the diagnosis of nasopharyngeal carcinoma (NPC) has attracted widespread attentions. Currently, NP brush sampling mostly relies on endoscopic guidance, and there are few reports on diagnostic markers suitable for nonguided conditions (blind brush sampling), which is of great significance for extending its application. One hundred seventy nasopharyngeal brushing samples were taken from 98 NPC patients and 72 non‐NPC controls under the guidance of endoscope, and 305 blind brushing samples were taken without endoscopic guidance from 164 NPC patients and 141 non‐NPC controls (divided into discovery and validation sets). Among these, 38 cases of NPC underwent both endoscopy‐guided NP brushing and blind brushing. EBV DNA load targeting BamHI‐W region and EBV DNA methylation targeting 11029 bp CpG site located at Cp‐promoter region were detected by quantitative polymerase chain reaction (q‐PCR). EBV DNA load showed good classification accuracy for NPC in endoscopy‐guided brushing samples (AUC = 0.984). However, in blind bushing samples, the diagnostic performance was greatly reduced (AUC = 0.865). Unlike EBV DNA load, the accuracy of EBV DNA methylation was less affected by brush sampling methods, whether in endoscopy‐guided brushing (AUC = 0.923) or blind brushing (AUC = 0.928 in discovery set and AUC = 0.902 in validation set). Importantly, EBV DNA methylation achieved a better diagnostic accuracy than EBV DNA load in blind brushing samples. Overall, detection of EBV DNA methylation with blind brush sampling shows great potential in the diagnosis of NPC and may facilitate its use in nonclinical screening of NPC. What's new? Detection of Epstein‐Barr virus (EBV) DNA load in nasopharyngeal (NP) brush samples is a promising diagnostic tool for nasopharyngeal carcinoma (NPC). Brush sampling, however, typically requires endoscopic guidance, whereas blind brushing, without endoscopy, could facilitate the use of brush sampling for NPC detection. Here, the authors compared methylation markers against EBV DNA load for NPC diagnosis in endoscopy‐guided and nonguided brush samples. Under blind brushing conditions, a qPCR‐based method for detecting EBV DNA methylation outperformed EBV DNA load detection for NPC diagnosis. Blind brushing, combined with EBV DNA methylation detection, could have significant advantages for NPC diagnosis in nonclinical settings.