An ultrastable, highly dense single-molecule assay ideal for observing protein–DNA interactions is demonstrated. Stable click tethered particle motion leverages next generation click-chemistry to achieve an ultrahigh density of surface tethered reporter particles, and has low non-specific interactions, is stable at elevated temperatures to at least 45 °C, and is compatible with Mg2+, an important ionic component of many regulatory protein–DNA interactions. Prepared samples remain stable, with little degradation, for >6 months in physiological buffers. These improvements enable the authors to study previously inaccessible sequence and temperature-dependent effects on DNA binding by the bacterial protein, histone-like nucleoid-structuring protein, a global transcriptional regulator found in Escherichia coli. This greatly improved assay can directly be translated to accelerate existing tethered particle-based, single-molecule biosensing applications.