Laser tweezers Raman spectroscopy technology is used to explore the accumulation of biodegradable plastic poly-β -hydroxybutyric acid (PHB) in Cupriavidus necator H16 at single- cell level. With ammonium sulfate (2 g/L) as nitrogen source, the fermentation of PHB is performed with 0%, 0.5%, 1.0%, 2.0% or 3.0% of fructose (mass fraction) and Raman spectra of single cell are acquired at different time of incubation respectively. Major results are as follows. 1) The fermentation performs ideal PHB product yield at 20 g/L of fructose. 2) At different fructose levels, C. necator H16 cells take almost the same steps to synthesize bio-macromolecule in the initial and early stages of fermentation, while higher fructose level extends the log phase of growth. 3) The dynamics of Raman intensity from peaks 782, 1574, 1656 and 1732 cm–1, which derive from RNA, DNA, proteins and PHB respectively, reveals that the cells show stronger Raman intensity of nucleic acid for longer time under higher level of fructose, and this may be of important factor affecting the accumulation of PHB. 4) The average intensity of single cell at peak 1732 cm- 1 shows linear relationship with yield of PHB and can quantitatively follow the PHB production. This observation uncovers the kinetics of major bio-macromolecule of C. necator H16 during the PHB accumulation at different fructose levels, provides novel spectral information for the PHB fermentation. This work shows the potential of laser tweezers Raman spectroscopy for the quick screening of high-yield PHB strains.