This study aimed to (i) investigate long interspersed nuclear element-1 (LINE-1) methylation levels of oral squamous cell carcinomas (OSCCs), the major type of oral malignancies; and (ii) investigate whether the hypomethylation of LINE-1s can be detected in oral rinses of OSCC patients. The combined bisulfite restriction analysis polymerase chain reaction (PCR) of LINE-1s (COBRALINE-1) was used. We found that tissues from OSCC specimens had lower methylation levels of LINE-1s than cells collected from the oral rinses of normal volunteers. Interestingly, cells collected from oral rinses of OSCC patients also revealed hypomethylated LINE-1s at the same level as OSCC tissues. There was no difference in the level of hypomethylation due to stages, locations, histological grades, and history of betel chewing, smoking and/or alcohol consumption. In conclusion, OSCCs possessed global hypomethylation and this alteration could be detected from oral rinses of OSCC patients by a simple PCR technique, COBRALINE-1. Therefore, COBRALINE-1 of oral rinses may be applied for non-invasive detection of oral malignancies.