The multidrug resistance of tumor cells has been creating a high requirement on development of nanocarriers for administration of different drugs, among which their drug loading capacity and controllable release properties are the key factors to overcome tumor drug resistance. This study aims to use a kind of bioactive 2D nanoplatelets (25 nm in diameter and 0.92 nm in thickness) to load different anticancer drugs (doxorubicin and methotrexate) via a step-by-step assembly, where variation of each drug amount can be used for adjustment of the sizes of the resulting nanocomplexes. The dual-drug loaded nanosystems allow for a sequential release of the loaded drugs. Furthermore, drug release rate can be accelerated under both acidic pathological triggers of tumors and/or by heating treatment, resulting in a synergistic anticancer bioactivity. The drug-mediated formation of nanocarriers may enlighten a design of novel nanoplatform for co-delivery of therapeutic agents, beyond anticancer drugs, in a combinative way for drug delivery applications.