microRNAs (miRNAs) have been intensively studied as valuable biomarkers in cardiometabolic disease. Typically, miRNAs are detected in plasma or serum but the use of samples collected in heparinized tubes is problematic for miRNA studies utilizing qPCR. Heparin and its derivatives interfere with qPCR-based analysis, leading to a substantial reduction or even complete loss of detectable miRNA levels. Given that red blood cells (RBCs) express abundant miRNAs, whose expression is altered in cardiometabolic disease, RBCs could serve as an attractive alternative in biomarker studies. Here, we aim to explore the stability of miRNAs in RBCs collected from whole blood with different anticoagulants and thereby the potential of RBCs as alternative materials for miRNA biomarker studies. miRNA profiling was performed in human RBCs via RNA sequencing, followed by qPCR validation of selected miRNAs in RBCs and plasma in both heparinized and EDTA tubes. RNA sequencing revealed abundant miRNA presence in RBCs isolated from blood collected in EDTA tubes. miR-210-3p, miR-21-5p, miR-16-5p, and miR-451a were detected at comparable levels in RBCs isolated from both heparinized and EDTA tubes, but not in plasma from heparinized tubes. Of note, miR-210 levels were consistently lower in RBCs from individuals with type 2 diabetes compared to healthy controls, regardless of anticoagulant type, supporting their potential as biomarker materials. In conclusion, RBCs offer a promising alternative for miRNA biomarker studies, overcoming heparin-related challenges.