Microalgae are a promising resource for biofuel production, although the lack of effective harvesting techniques limits their industrial use. In this context, flotation, and in particular dissolved air flotation (DAF), is an interesting separation technique that could drastically reduce harvesting costs and make biofuel-production systems more economically viable. But because of the repulsive interaction between cells and bubbles in water, the efficiency of this technique can be limited. To solve this problem, we propose here an original DAF process where bubbles are functionalized with a bio-sourced polymer able to specifically bind to the surface of cells, chitosan. In a first part, we modify chitosan by adding hydrophobic groups on its backbone to obtain an amphiphilic molecule, PO-chitosan, able to assemble at the surface of bubbles. Then, using a recently developed technique based on atomic force microscopy (AFM) combined with microfluidics, we probe the interactions between PO-chitosan coated bubbles and cells at the molecular scale; results show an enhanced adhesion of functionalized bubbles to cells (from 3.5 to 12.8 nN) that is pH-dependent. Separation efficiencies obtained in flotation experiments with functionalized bubbles are in line with AFM data, and a microalgae separation efficiency of approximately 60% could be reached in a single step. In addition, we also found that PO-chitosan could be used efficiently as a flocculant (nearly 100% of cells removed), and in this case AFM experiments revealed that the flocculation mechanism is based on hydrophobic interactions between cells and PO-chitosan. Altogether, this comprehensive study shows the interest of PO-chitosan to harvest cells in flotation or flocculation/flotation processes.