We present a droplet digital Enzyme-Linked Oligonucleotide Hybridization Assay (droplet digital ELOHA) for reference-free absolute quantification of nucleic acids. Droplet digital ELOHA is able to accurately quantify as few as several hundred copies of nucleic acids in a large, mL-sized sample volume (equivalent to 1 aM or lower) without molecular replication. Droplet digital ELOHA provides a linear response to the number of copies present in a sample to allow for detecting a small fold change unattainable by real time quantitative PCR. Its capability to cope with a large sample volume benefits from the use of magnetic beads to streamline capturing of nucleic acids from the sample and subsequent surface hybridization reactions in droplets. Droplet digital ELOHA is particularly attractive for RNA quantification since it does not require reverse transcription. The absolute quantification ability of droplet digital ELOHA is demonstrated with the analysis of clinical Neisseria Gonorrhoeae 16S rRNA to show its potential diagnostic value.