Many colorimetric biosensors utilise citrate-capped gold nanoparticles (AuNPcit) in combination with functional macromolecules. Proper function of the utilised macromolecules is strongly dependent on the buffer systems. However, it is well known that solvents and buffers might cause aggregation of nanoparticles. A comprehensive and systematic investigation on the effect of buffer composition, concentration and pH on the aggregation of AuNPcit is reported in this study. Distinct aggregation behaviours were observed in acidic and basic pH. In acidic pH, the increase in pH, caused stabilisation of AuNPcit, while in basic pH, the stability was dependent on the ionisation degree of the applied buffer. Theoretical analyses revealed that ionic buffer species act as citrate competitors and control aggregation of AuNPcit. Understanding the fundamental principles of competition between citrate and buffer components allows scientists to choose orthogonal conditions for development of gold nanoparticle-based biosensors which guarantee stability of gold nanoparticles and proper folding of macromolecules simultaneously.