A modified enzyme fragment complementation assay has been designed and validated as a turn-on biosensor for nucleic acid detection in dilute aqueous solution. The assay is target sequence-agonistic and uses fragments of NanoBiT, the split luciferase reporter enzyme, that are esterified enzymatically at their C-termini to steramers, sterol-linked oligonucleotides. The Drosophila hedgehog autoprocessing domain, DHhC, serves as the self-cleaving enzyme for the NanoBiT-steramer bioconjugations. Unlike current approaches, the final bioconjugate generated by DHhC and used for nucleic acid detection is free of self-labeling passenger protein. In the presence of single stranded (ss) DNA or RNA template with adjacent segments complementary to the Nano-BiT steramer oligonucleotides, the two NanoBiT fragments associate productively, reconstituting NanoBiT's luciferase activity. In samples containing ssDNA or RNA template at low nM concentrations, NanoBiT luminescence exceeded background signal by 30- to 60-fold. The steramer probe sequences used to prepare these sensors are unconstrained in length and composition. In the absence of sequence constraints of the probe element and without the added bulk of a self-labeling protein, these NanoBiT-steramer bioconjugates open new applications in the programmable detection of small fragments of coding and noncoding DNA and RNA.