This article reports a high-yield procedure for preparing microsized (giant) Janus liposomes via gel-assisted lipid swelling and clustering behavior of these liposomes directed by biotin-avidin affinity binding. Confocal fluorescence microscopy reveals in detail that these new lipid colloidal particles display broken symmetry and heterogeneous surface chemistry similar to other types of Janus particles. An optimized formation procedure is presented, which reproducibly yields large liposome populations dominated by a single-domain configuration. This work further demonstrates that biotin-conjugated 1,2-dioleoyl-sn-glycero-3-phosphoetha-nolamine preferentially partitions into the liquid-disordered phase of the lipid matrix, rendering these Janus liposomes asymmetrical binding capacity toward avidin. This affinity binding drives irreversible and domain-specific cluster formation among Janus liposomes, whose structure and size are found to depend on the domain configuration of individual liposomes and incubation time.