Calixarene is the third generation of supra-molecular compounds after crown ether and cyclodextrin. Amphiphilic calixarene can be obtained by modulation with both hydrophilic group and hydrophobic alkyl chain. Compared with conventional surfactant, amphiphilic calixarene has much lower critical micelle concentration and is much easier to self-assemble into different morphological aggregates. Calixarene-basedsupra-amphiphile can be designed via noncovalent bonds due to the capability of calixarene to recognize surfactant; the binding of a surfactant with calixarene can decrease the critical micelle concentration of surfactant by several times. The calixarene-surfactant complex can self-aggregate to form spherical micelles, vesicles, and spherical nanoparticles, and the aggregation behavior can be controlled by the structures and the molar ratio of surfactant to calixarene and environmental factors. Calixarene-based amphiphile and supra-amphiphile show low cytotoxicity. They can load drugs and assemble into nanocapsules with drugs. The structure of the calixarene-drug complex can respond to external stimuli, rendering the sustained release of the drug and suggesting its potential application as a drug delivery system. Recently, calixarene has also been found to selectively bind proteins, suggesting its prospect in disease diagnosis and intervention treatment in clinics. This review elaborates on the research progress in the self-assembly behaviors of calixarene-based amphiphile and supra-amphiphile and the applications of the calixarenes in drug delivery and protein recognition. The prospectives for the studies are also provided in this review.