The direct production of macromolecular scale (sub-5 nm) porous nanocrystals with high surface area has been a considerable challenge over the past two decades. Here we report an interfacial site-directed capping agent-free growth method to directly produce porous ultrasmall (sub-5 nm), fully crystalline, macromolecular scale nanocrystals. The porous sub-5 nm Prussian blue nanocrystals exhibit uniform sizes (∼4 ± 1 nm), high surface area (∼855 m2 g-1), fast electron transfer (rate constant of ∼9.73 s-1), and outstanding sustained catalytic activity (more than 450 days). The nanocrystal-based biointerfaces enable unprecedented sub-nanomolar level recognition of hydrogen peroxide (∼0.5 nM limit of detection). This method also paves the way towards the creation of ultrasmall porous nanocrystals for efficient biocatalysis.