We developed DNA-spider silk conjugates using click chemistry, combining short DNA strands with the recombinant protein eADF4(C16), which we utilized to construct thin nanofibril-based coatings demonstrating swelling and softening properties of nanohydrogels. Exploiting the DNA-functionalization and cell-repellent attributes of the fibrillar networks, we devised specific cell immobilization strategies. Introducing a mild lipid-DNA modification to cell membranes we achieved highly specific DNA-assisted immobilization of cells, allowing for tunable cell densities. Moreover, we used nanohydrogels containing aptamers to selectively bind to cell markers, immobilizing various types of cancer cells. We integrated the principles of the surface-nucleated nanohydrogel formation with photolithography, which enabled formation of microwells of arbitrary shapes dedicated to formation of DNA-functionalized micropatterned arrays, enabling both, the DNA-assisted immobilization of DNA-labelled cells and the aptamer-cancer cell marker interactions with high spatial fidelity.