Analysis of the characteristics of biomolecules, including size, charge and binding kinetics, is essential for biomedical and life science research and applications. State-of-the-art protein analysis methods rely on separate technologies to quantify these characteristics, and considerable time, cost and analytes are required. Lack of single molecule analysis capability in above methods also making them difficult to study heterogeneous processes and achieving precision diagnosis.To address these issues, several techniques based on surface sensitive optical imaging principles were developed. The first technique is evanescent scattering microscopy (ESM) with single molecule resolution, which is capable of imaging single immunoglobulin G with high signal-to-noise ratio. In addition, nano-oscillator was combined with the ESM to achieve the simultaneous size and charge detection of single proteins. Based on the unique high axial sensitivity of the surface plasmon resonance (SPR), a 3D tracking technique to study the motion and interaction of biomolecules was introduced. With the additional dimension, more information in particle motions can be revealed compared to conventional 2D bright field tracking. By tracking the motion of nanoparticles, motion pattern of tethered nanoparticles and interaction between double-stranded DNA and an enzyme can be visualized. The G protein-coupled receptors (GPCRs) expressed virion oscillator array for quantification of the binding kinetics of small molecule drugs and different GPCRs was attempted. Cross-talking signals between the array spots were discovered, and several control experiments were performed to explore the possible reason. As an alternative solution for multiplexing, DNA barcode technique was implemented with the GPCR virions and achieved with the ESM, which paved a way for multiplexed single molecule binding kinetics studies. Circular RNAs has been found as an important class of regulators at the transcriptional and posttranscriptional level and could be potential biomarkers for many diseases. However, determination of its existence from the linear RNAs is challenging for the tradition molecular detection methods. Due to the no ending feature, by designing a unique complementary probe sequence, hybridization affinity difference between circular and linear RNA can be distinguished. Affinities with different hybridization nucleotides number were measured and verified.