Measurement in liquid media is a major challenge in real-time detection using resonant cantilevers. This is addressed in the present study by fabricating sub-micron thick cantilevers followed by functionalization for biomolecule detection. The fabricated cantilever resonator beams of thickness 165 nm were used for measurements in two systems: (i) human immunoglobulin (HIgG) as the antibody on the cantilever sensing mouse immunoglobulin (MIgG) as corresponding antigen, and (ii) detection of triglyceride (TG) based on the enzymatic hydrolysis with lipase, using tributyrin as a model. In both cases, the beams were functionalized for covalent bonding of the protein receptor. The label-free detection was carried out by measuring the shift in resonance frequency at higher modes, using a laser Doppler vibrometer in liquid and in air. The calibration showed a linear correlation between the bioanalyte concentration and change in the resonance frequency. Notably, detection of antigen mass as low as 434 ± 59fg and triglyceride concentration in the nM range with limit of detection as 7 nM in liquid interface was achieved, greatly improving the sensitivity of bioanalyte detection in liquid samples. Although frequency-based methods are highly sensitive, the issues with measurement liquid medium limit their application. In the present report, these issues were addressed by fabricating sub-micron thick cantilever beam, choosing an appropriate functionalization method without affecting the sensitivity, and measurement at higher modes. These have resulted in circumventing issues like damping and hydrodynamic loading thus improving its potential as real-time sensor. © 2020 IOP Publishing Ltd.