Highlights • Reliable detection and quantification of myoglobin in blood plasma is realized through an upconversion nanoparticles based lateral flow assay. • The assay is able to reach a limit of detection as low as 0.21 ng/mL, a linear range of 10–400 ng/mL, and a detection time within 10 min. • The assay is commensurate with the clinically used Abbott Chemiluminescence system on Mb concentration evaluation in human blood plasmas. Abstract Reliable detection and quantification of myoglobin (Mb) in blood plasma is of significance for clinical diagnosis and management of acute myocardial infarction (AMI). Here, we describe a homemade platform combining efficient core-shell upconversion nanoparticles (UCNPs) and lateral flow assay (LFA) for ultrasensitive and specific fluorescence ratiometric evaluation of Mb in clinical blood samples. After a systematic optimization, this platform was able to reach a limit of detection as low as 0.21 ng/ml, a linear range of 0.5–400 ng/ml, a recovery rate of 90.6–110.5%, an inter-assay coefficient of variation (CV) of 3.0–6.2%, an intra-assay CV of 8.8–13.6%, and a detection time of 10 min. Furthermore, the acquired concentration results were unresponsive to the interference of high-bilirubin, high-lipids, and hemolysis, and a range of other biomarkers (CRP, PCT, and BSA), substantiating robust Mb-specific detection. Importantly, the platform showed a significant linear correlation (R2 = 0.9484) with clinically used Abbott Chemiluminescence detection system (CLDS) on evaluation of Mb concentration in 50 clinical human blood plasmas. The achievement of rapid, sensitive and reliable quantification of Mb in clinical plasma promises the prevalent use of the described platform for on-field diagnosis or rule-out of AMI in clinical practice. [ABSTRACT FROM AUTHOR]