A cheap and simple procedure for the simultaneous colorimetric determination of glycerol, magnesium and calcium in biodiesel samples using a paper-based analytical device (PAD) was developed. A reversed-phase air-assisted dispersive liquid-liquid microextraction (RP-AA-DLLME) approach was proposed and used for glycerol, magnesium and calcium separation from biodiesel samples into aqueous phase (sodium periodate solution). After RP-AA-DLLME the obtained hydrophilic emulsion (biodiesel fuel in water) containing target analytes was introduced to a sample zone of the PAD, and as the emulsion penetrated through hydrophilic cellulose fibers to detection zones its destruction was observed. Colorimetric detection of calcium and magnesium with Eriochrome Black T and derivative of glycerol (formaldehyde) with acetylacetone on the detection zones of the PAD was implemented using conventional scanner and Corel® PHOTO-PAINT™ X7 software. Under optimal conditions, the calibration curves were linear in the ranges 100–300 μg g^{− 1} for glycerol and 1–15 μg g^{− 1} for calcium and magnesium. The limits of detection, calculated from a blank test based on 3σ, were 30 μg g^{− 1} for glycerol and 0.3 μg g^{− 1} for calcium and magnesium. The PAD was successfully applied for biodiesel samples analysis. Novelty statement: A first paper-based analytical device for the simultaneous colorimetric determination of glycerol, calcium and magnesium in biodiesel samples was successfully developed. A novel approach for biodiesel samples pretreatment based on a reversed-phase air-assisted dispersive liquid-liquid microextraction was also proposed for the first time. The reversed-phase air-assisted dispersive liquid-liquid microextraction was coupled with paper-based analytical device and applied for the simultaneous colorimetric determination of glycerol, calcium and magnesium in biodiesel samples. In this study, it was found, that glycerol extraction kinetics from biodiesel samples to aqueous phase can be accelerated in the presence of periodate-ions due to analyte oxidation to water-soluble formaldehyde. This feature allowed improving glycerol extraction efficiency. The proposed procedure has several advantages including miniaturization, cost effectiveness and simplicity.