Microfluidic sampling media based on paper and its modifications with either gold nanoparticles or sputtered gold were evaluated for potentiometric determination of Na+, K+, and Cl– ions in clinically relevant samples. The measurements were conducted in comparison to other commonly considered microfluidic substrates, i.e. sponge, polyester textile, and polyamide textile. Ion determination was done by using solid-contact ion-selective electrodes based on plasticized PVC membranes for Na+, K+, and Cl– ions and utilizing PEDOT(PSS) or PEDOT(Cl) as the ion-to-electron transducer. The solid-contact ion-selective electrodes and a solid-state reference electrode were placed directly on the substrate into which the sample solution was wicked. Transport of bovine serum albumin (BSA) through the paper substrate was studied by ellipsometry. Modification of the paper substrates by gold nanoparticles (AuNPs) was found to slow down the transport of BSA through the paper, when compared with unmodified paper substrates and when compared with all the other alternative sampling matrices studied. The retention of BSA obtained with AuNP-modified paper substrates significantly improved the accuracy of the potentiometric ion determinations in sweat, saliva, artificial tears, and artificial serum. The potentiometric results were validated by inductively coupled plasma optical emission spectrometry (ICP-OES) and ion chromatography (IC). The study indicates that modification of paper by AuNPs is a feasible approach to reduce biofouling of sensors that are used in the paper-based analysis of clinically relevant samples.