This paper presents a novel cylindrical centrifuge designed to separate microspheres and cells. The polyvinylidene-fluoride tubes with heat shrink characteristics were used to encapsulate helix microchannels. Numerical simulations were employed to analyze and design the channel helix structure (with five annular loops, 2 mm pitch and 20 mm cylinder diameter). Then, The inertial focusing of microspheres (with diameters of 10, 15 and 20 µm) and the freshwater microalgae (i.e. Haematococcus pluvialis) in the cylindrical centrifuge were studied experimentally. As the particle size increased, the focusing position moved toward the center line of the flow channel, and the focus degree tended to decrease. When the flow rate increased, the focus position barely changed, but the focus degree increased significantly. A quantitative study of the centrifuge efficiency revealed that when the initial concentration was 104 particles ml−1 and the flow rate was at its optimal 1.7 ml min−1, the centrifugal efficiency (CE) values of 10 µm, 15 µm, 20 µm microspheres, and H. pluvialis, were 98.8%, 87.8%, 70.8% and 64.6%, respectively. The CE is inversely proportional to the microsphere size and the initial concentrations. Compared with the other two centrifuges (the cavity-vortex and planar spiral), the cylindrical centrifuge design and manufacturing process have a simplicity that provides low cost, efficient sample handling, and effective separation of microspheres and biological cells.