Electrospray scanning mobility particle sizer (ES-SMPS) is an electrophoretic method for size characterization of nanoparticles (NPs) and its population in biological samples such as macromolecules and virus is growing. To ensure the measurement precision and accuracy, especially for the sub-10 nm measurement, size calibration of ES-SMPS system is necessary. Gold nanoparticles (AuNPs) are commonly chosen as the reference materials (RMs) while the surfactants preventing NP aggregate coat among NP surface lead to the measurement uncertainty in ES-SMPS. In this paper, both AuNPs and self-disperse biological macromolecules, bio-NPs, were selected for the evaluation of their suitability as RMs for ES-SMPS. Two methods including centrifugation and heating were used for the surfactant removal from AuNPs suspension while the results showed little influence of the centrifugal and thermal treatments on the accuracy improvement of AuNPs, where more than 22% difference from the nominal diameter remained in AuNPs. In the case of bio-NPs, bovine serum albumin (BSA), ubiquitin, low-density lipoprotein (LDL) and high-density lipoprotein(HDL) were suspended in electrolyte. BSA and ubiquitin presented good disperse and repeatable size compared to literature, high stability at time intervals, and consequently were suggested as ideal sub-10 nm RM for ES-SMPS calibration.