Aims A scanning laser ophthalmoscope (SLO) has been used for multifocal electroretinography (mf ERG) measurements under simultaneous fundus monitoring. The aim of this study was to prove if the SLOmfERG measurement reflects reliably the clinically registered underlying disease, and to demonstrate the importance of its main advantage, fixation monitoring. Methods In all, 10 patients with macular hole stage II/III were included in the study, and 19 normal individuals served as the control group. The mf ERG device was combined with an SLO, which was used both as a stimulus and trigger unit as well as a fundusmonitoring system. Monitoring of the fundus was guaranteed by an infrared laser (780 nm). The stimulus matrix consisted of 61 hexagonal elements, covering 241 of the posterior pole. We examined both, patients with macular holes and healthy individuals. Results Compared to normal controls, patients with a macular hole (Gass stage III) showed a significant decrease in response density in the centre of the stimulus array, which correlated well with the morphological alteration observed by clinical examination. However, variation of response density of the central hexagonal area has been proved to be high. Conclusions SLO-mfERG is a feasible and reliable new technique to investigate macular function under simultaneous fundus control. The main advantage is that control of fixation can be used in order to obtain more reliable results that correlate well with visible fundus abnormalities such as in patients with macular holes. However, further investigations have to be performed in order to overcome sufficiently the problem of fixation instability.